U.S. patent application number 17/602511 was filed with the patent office on 2022-05-26 for antibody to tigit and use thereof.
The applicant listed for this patent is GREEN CROSS CORPORATION, MOGAM INSTITUTE FOR BIOMEDICAL RESEARCH. Invention is credited to Min Kyu HUR, Mun Kyung KIM, Eun Hee LEE, Jee Won LEE, Ok Jae LIM, So Jung LIM, Yang Mi LIM, Hye Mi NAM, Hye-Young PARK, Joong Hyuk SHEEN, Eun Jung SONG, Jong Hwa WON, Hye In YUM.
Application Number | 20220162310 17/602511 |
Document ID | / |
Family ID | 1000006171316 |
Filed Date | 2022-05-26 |
United States Patent
Application |
20220162310 |
Kind Code |
A1 |
PARK; Hye-Young ; et
al. |
May 26, 2022 |
ANTIBODY TO TIGIT AND USE THEREOF
Abstract
The present invention relates to: an antibody to T cell
Immunoreceptor with Ig and Tyrosine-Based Inhibitory Motif Domains
(TIGIT), or an antigen-binding fragment thereof; a nucleic acid
encoding same; a vector carrying the nucleic acid; a cell
transformed with the vector; a method for producing the antibody or
the antigen-binding fragment thereof; and a composition and a
composition for combined administration, which comprise same and
are for preventing or treating cancer.
Inventors: |
PARK; Hye-Young;
(Gyeonggi-do, KR) ; SONG; Eun Jung; (Gyeonggi-do,
KR) ; LEE; Eun Hee; (Gyeonggi-do, KR) ; YUM;
Hye In; (Gyeonggi-do, KR) ; NAM; Hye Mi;
(Gyeonggi-do, KR) ; KIM; Mun Kyung; (Gyeonggi-do,
KR) ; LEE; Jee Won; (Gyeonggi-do, KR) ; SHEEN;
Joong Hyuk; (Gyeonggi-do, KR) ; HUR; Min Kyu;
(Gyeonggi-do, KR) ; LIM; So Jung; (Gyeonggi-do,
KR) ; LIM; Ok Jae; (Gyeonggi-do, KR) ; LIM;
Yang Mi; (Gyeonggi-do, KR) ; WON; Jong Hwa;
(Gyeonggi-do, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GREEN CROSS CORPORATION
MOGAM INSTITUTE FOR BIOMEDICAL RESEARCH |
Gyeonggi-do
Gyeonggi-do |
|
KR
KR |
|
|
Family ID: |
1000006171316 |
Appl. No.: |
17/602511 |
Filed: |
May 22, 2020 |
PCT Filed: |
May 22, 2020 |
PCT NO: |
PCT/KR2020/006705 |
371 Date: |
October 8, 2021 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 45/06 20130101;
A61P 35/00 20180101; C07K 2317/565 20130101; C07K 16/2818 20130101;
C07K 2317/92 20130101; C07K 16/2803 20130101; A61K 2039/505
20130101; C07K 2317/76 20130101; A61K 39/3955 20130101; C07K
2317/33 20130101; C07K 2317/622 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61P 35/00 20060101 A61P035/00; A61K 45/06 20060101
A61K045/06; A61K 39/395 20060101 A61K039/395 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 13, 2019 |
KR |
10-2019-0069931 |
Claims
1. An antibody specifically binding to TIGIT (T-cell immunoreceptor
with Ig and tyrosine-based inhibitory motif domains) or an
antigen-binding fragment thereof, comprising: a heavy-chain CDR1
represented by SEQ ID NO: 3, 6, 9 or 12; a heavy-chain CDR2
represented by SEQ ID NO: 4, 7 or 10; a heavy-chain CDR3
represented by SEQ ID NO: 5, 8, 11 or 13; a light-chain CDR1
represented by SEQ ID NO: 14, 17, 20, 23 or 25; a light-chain CDR2
represented by SEQ ID NO: 15, 18, 21 or 26; and a light-chain CDR3
represented by SEQ ID NO: 16, 19, 22, 24 or 27.
2. The antibody or antigen-binding fragment thereof according to
claim 1, wherein the antibody or antigen-binding fragment thereof
comprises: a heavy-chain CDR1 of SEQ ID NO: 3, a heavy-chain CDR2
of SEQ ID NO: 4, a heavy-chain CDR3 of SEQ ID NO: 5, a light-chain
CDR1 of SEQ ID NO: 14, a light-chain CDR2 of SEQ ID NO: 15, and a
light-chain CDR3 of SEQ ID NO: 16; a heavy-chain CDR1 of SEQ ID NO:
6, a heavy-chain CDR2 of SEQ ID NO: 7, a heavy-chain CDR3 of SEQ ID
NO: 8, a light-chain CDR1 of SEQ ID NO: 17, a light-chain CDR2 of
SEQ ID NO: 18, and a light-chain CDR3 of SEQ ID NO: 19; a
heavy-chain CDR1 of SEQ ID NO: 9, a heavy-chain CDR2 of SEQ ID NO:
10, a heavy-chain CDR3 of SEQ ID NO: 11, a light-chain CDR1 of SEQ
ID NO: 20, a light-chain CDR2 of SEQ ID NO: 21, and a light-chain
CDR3 of SEQ ID NO: 22; a heavy-chain CDR1 of SEQ ID NO: 9, a
heavy-chain CDR2 of SEQ ID NO: 10, a heavy-chain CDR3 of SEQ ID NO:
11, a light-chain CDR1 of SEQ ID NO: 23, a light-chain CDR2 of SEQ
ID NO: 21, and a light-chain CDR3 of SEQ ID NO: 24; or a
heavy-chain CDR1 of SEQ ID NO: 12, a heavy-chain CDR2 of SEQ ID NO:
10, a heavy-chain CDR3 of SEQ ID NO: 13, a light-chain CDR1 of SEQ
ID NO: 25, a light-chain CDR2 of SEQ ID NO: 26, and a light-chain
CDR3 of SEQ ID NO: 27.
3. The antibody or antigen-binding fragment thereof according to
claim 1, wherein the antibody or antigen-binding fragment thereof
comprises at least one heavy-chain variable region selected from
the group consisting of SEQ ID NOS: 28 to 32.
4. The antibody or antigen-binding fragment thereof according to
claim 1, wherein the antibody or antigen-binding fragment thereof
comprises at least one light-chain variable region selected from
the group consisting of SEQ ID NOS: 34 to 38.
5. A nucleic acid encoding the antibody or antigen-binding fragment
thereof according to claim 1.
6. A vector comprising the nucleic acid according to claim 5.
7. A cell transformed with the vector according to claim 6.
8. A method of producing an antigen binding to TIGIT or an
antigen-binding fragment thereof comprising: (a) culturing the cell
according to claim 7; and (b) recovering an antibody or
antigen-binding fragment thereof from the cultured cell.
9. A composition for preventing or treating cancer comprising the
antibody or antigen-binding fragment thereof according to claim 1
as an active ingredient.
10. A composition for preventing or treating cancer using the
antibody or antigen-binding fragment thereof according to claim 1
in combination with an anticancer therapy.
11. A composition for preventing or treating cancer using the
antibody or antigen-binding fragment thereof according to claim 1
in combination with an immune checkpoint inhibitor.
12. The composition according to claim 11, wherein the immune
checkpoint inhibitor targets PD-1.
Description
TECHNICAL FIELD
[0001] The present invention relates to an antibody that
specifically binds to TIGIT (T-cell immunoreceptor with Ig and
tyrosine-based inhibitory motif domains), an antigen-binding
fragment thereof, a nucleic acid encoding the same, a vector
comprising the nucleic acid, a cell transformed with the vector, a
method of producing the antigen and antigen-binding fragment
thereof, a composition for preventing or treating cancer comprising
the same, and a composition for preventing or treating cancer
comprising the same for administration in combination with another
therapeutic agent.
BACKGROUND ART
[0002] There are various cases involving cancer tissues,
specifically, immune cells may be incapable of accessing cancer
tissues, or immune cells may be capable of penetrating into the
cancer tissues but the immune response may be suppressed by the
cancer tissues. The human immune system has an immune checkpoint
system to suppress a hyperimmune response caused by excessive
proliferation of T-cells. An immune checkpoint blockade targeting
an immune checkpoint protein involved in the immune checkpoint does
not directly target cancer cells, but causes CTL (cytotoxic T
cells) to eliminate cancer cells by restoring the activity of TIL
(tumor-infiltrating lymphocytes), in particular, CTL, which are
located around the cancer tissues, but the activity thereof is
reduced by immunosuppressive factors (CD80, TIGIT) expressed by
cancer cells. In this case, the immune checkpoint blockade blocks
inhibitory signaling by preventing binding between T-cell
inhibition receptors and inhibitory factors on the surface of
cancer cells, and enables effective other stimulatory signaling,
thus ultimately having an effect of increasing the activity of
CTL.
[0003] Administration of a large number of conventional
chemotherapeutic agents inevitably should be stopped due to the
severe side effects thereof. However, immune checkpoint blockers
exhibit clinical superiority in terms of overall survival,
progression-free survival, and the like, and cause only mild side
effects. In addition, conventional chemotherapeutic agents cannot
be administered when the same cancer recurs because of acquired
resistance to the therapeutic agent, whereas an anticancer immune
response due to the administration of an immune checkpoint blocker
causes a memory response upon recurrence of the same cancer,
enabling rapid eradication of cancer cells.
[0004] Regarding the immune checkpoint blocker, ipilimumab, which
is a monoclonal antibody specific for the immune checkpoint
receptor CTLA-4 (cytotoxic T-lymphocyte associated antigen-4), has
shown effectiveness in treating metastatic malignant melanoma. In
addition, monoclonal antibodies specific for PD-1 (programmed cell
death-1) and PD-L1 (programmed death ligand-1), which is a ligand
for PD-1, are being developed. Representative examples thereof
include nivolumab, pembrolizumab, avelumab, atezolizumab,
durvalumab and the like. The effects of PD-1 or PD-L1 inhibitors
are found in various tumors as well as in malignant melanoma.
[0005] Meanwhile, TIGIT (=Vstm-3, WUCAM) is a co-inhibitory
molecule whose expression is induced when NK cells and T cells are
activated, like PD-1. Cancer cells can escape attack from T cells
by inhibiting T cells by overexpressing ligands for TIGIT. An
anti-TIGIT antibody is predicted to prevent cancer progression by
restoring the immune function of cancer patients by blocking the
suppression of immune activity through TIGIT present in cancer
cells.
[0006] TIGIT is induced and expressed in CD8+CTL and CD4+ T helper
cells, similar to PD-1, and is continuously expressed along with
PD-1 in FoxP3+ Treg, which plays a leading role in suppressing the
immune response in cancer tissues, and is induced and expressed in
higher levels when Treg is activated.
[0007] Several types of anti-TIGIT antibodies have been reported to
date (US Patent Application Publication No. 2017-0088613, etc.),
but research on specific mechanisms is still insufficient, and
antibodies having efficacy to an extent enabling use as actual
therapeutic agents have not been developed. So, demand for a
TIGIT-specific antibody having high efficacy is still
increasing.
[0008] Against this technical background, the present inventors
endeavored to develop antibodies that specifically bind to TIGIT.
As a result, the present inventors developed an anti-TIGIT antibody
that binds to TIGIT with high affinity, and found that this
anti-TIGIT antibody acts as an effective immuno-oncology agent.
Based thereon, the present invention has been completed.
SUMMARY OF THE INVENTION
[0009] It is one object of the present invention to provide a novel
antibody to TIGIT or an antigen-binding fragment thereof.
[0010] It is another object of the present invention to provide a
nucleic acid encoding the antibody or an antigen-binding fragment
thereof.
[0011] It is another object of the present invention to provide a
vector comprising the nucleic acid, a cell transformed with the
vector, and a method for producing the same.
[0012] It is another object of the present invention to provide a
composition for preventing or treating cancer comprising the
antibody or an antigen-binding fragment thereof.
[0013] It is another object of the present invention to provide a
composition for preventing or treating cancer by administering the
antibody or an antigen-binding fragment thereof in combination with
another drug.
[0014] To achieve the above objects, the present invention provides
an antibody or an antigen-binding fragment thereof specifically
binding to TIGIT (T-cell immunoreceptor with Ig and tyrosine-based
inhibitory motif domains) comprising a heavy-chain CDR1 represented
by SEQ ID NO: 3, 6, 9 or 12, a heavy-chain CDR2 represented by SEQ
ID NO: 4, 7 or 10, a heavy-chain CDR3 represented by SEQ ID NO: 5,
8, 11 or 13, a light-chain CDR1 represented by SEQ ID NO: 14, 17,
20, 23 or 25, a light-chain CDR2 represented by SEQ ID NO: 15, 18,
21 or 26, and a light-chain CDR3 represented by SEQ ID NO: 16, 19,
22, 24 or 27.
[0015] The present invention also provides a nucleic acid encoding
the antibody or an antigen-binding fragment thereof.
[0016] The present invention also provides a vector comprising the
nucleic acid.
[0017] The present invention also provides a cell transformed with
the vector.
[0018] The present invention also provides a method of producing
the antigen or antigen-binding fragment thereof comprising (a)
culturing the cell and (b) recovering an antibody or
antigen-binding fragment thereof from the cultured cell.
[0019] The present invention also provides a composition for
preventing or treating cancer comprising the antibody or
antigen-binding fragment thereof as an active ingredient.
[0020] The present invention also provides a composition for
preventing or treating cancer using the antibody or antigen-binding
fragment thereof in combination with another anticancer
therapy.
BRIEF DESCRIPTION OF DRAWINGS
[0021] FIG. 1 shows the result of a test on the binding capacity of
a scFv-form anti-TIGIT antibody.
[0022] FIG. 2 shows the result of a test on the binding capacity of
an IgG-type anti-TIGIT antibody.
[0023] FIG. 3 shows the results of a blocking assay for TIGIT and
the ligand thereof, poliovirus receptor (PVR), performed on 11
anti-TIGIT antibodies.
[0024] FIG. 4 shows the affinity of the anti-TIGIT antibody,
measured by Octet.
[0025] FIG. 5 shows the affinity of the anti-TIGIT antibody
measured by BIACORE.
[0026] FIG. 6 shows the binding capacity between TIGIT expressed on
the cell surface and TIGIT candidate antibodies.
[0027] FIG. 7 shows the competitiveness of the TIGIT candidate
antibody to PVR, which is a ligand of TIGIT.
[0028] FIG. 8 shows the result of an assay for T-cell activation
due to the binding inhibition effect between TIGIT and PVR by the
TIGIT candidate antibody.
[0029] FIG. 9 shows the results of a cross-reaction to determine
whether or not TIGIT candidate antibodies are capable of binding to
TIGIT in mice, monkeys and humans.
[0030] FIG. 10 shows the result of confirmation that the function
of the regulatory T cells is inhibited by the TIGIT candidate
antibody, using co-culture of regulatory T cells and CFSE-labeled
responder cells.
[0031] FIG. 11 shows the result of confirming the anticancer effect
of the TIGIT candidate antibody.
[0032] FIGS. 12a and 12b show results of analysis of changes in
T-cell proliferation and IFN-.gamma. secretion when treated with
the specified antibodies in a combined lymphocyte reaction using
dendritic cells and allogeneic T cells.
[0033] FIG. 13 shows the result of analyzing the proportion of
cells expressing each activation marker upon treatment of the
regulatory T cells of a healthy donor with the antibody
specified.
[0034] FIG. 14 shows the result of analyzing the proportion of
cells expressing exhaustion markers in CD8-positive T cells and
regulatory T cells of a multiple myeloma patient.
[0035] FIG. 15 shows the result of analyzing the change in
IFN-.gamma. secretion upon treatment of PBMCs from a multiple
myeloma patient with the specified antibody.
[0036] FIG. 16 shows the result of analyzing the expression of PVR
in the HT29 cell line.
DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS OF THE INVENTION
[0037] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as those appreciated by those
skilled in the field to which the present invention pertains. In
general, the nomenclature used herein is well-known in the art and
is ordinarily used.
[0038] In one aspect, the present invention is directed to an
antibody or an antigen-binding fragment thereof specifically
binding to TIGIT (T-cell immunoreceptor with Ig and tyrosine-based
inhibitory motif domains), wherein the antibody or an
antigen-binding fragment thereof comprising a heavy-chain CDR1
represented by SEQ ID NO: 3, 6, 9 or 12, a heavy-chain CDR2
represented by SEQ ID NO: 4, 7 or 10, a heavy-chain CDR3
represented by SEQ ID NO: 5, 8, 11 or 13, a light-chain CDR1
represented by SEQ ID NO: 14, 17, 20, 23 or 25, a light-chain CDR2
represented by SEQ ID NO: 15, 18, 21 or 26, and a light-chain CDR3
represented by SEQ ID NO: 16, 19, 22, 24 or 27.
[0039] The TIGIT comprises the sequence of SEQ ID NO: 1.
TABLE-US-00001 Human TIGIT (SEQ ID NO: 1)
MGWCLLLIWAQGLRQAPLASGMMTGTIETTGNISAEKGGSIILQCHLSST
TAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTV
NDAGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIPLLGAMAATL
VVICTAVIVVVALTRKKKALRIHSVEGDLRRKSAGQEEWSPSAPSPPGSC
VQAEAAPAGLCGEQRGEDCAELHDYFNVLSYRSLGNCSFFTETG
[0040] As used herein, the term "antibody" refers to an anti-TIGIT
antibody that specifically binds to TIGIT. The scope of the present
invention comprises not only a complete antibody specifically
binding to TIGIT but also an antigen-binding fragment of the
antibody molecule.
[0041] The antibody of the present invention comprises, but is not
limited to, monoclonal antibodies, multispecific antibodies, human
antibodies, humanized antibodies, chimeric antibodies, single-chain
Fvs (scFVs), single-chain antibodies, Fab fragments, F(ab')
fragments, disulfide-bond Fvs (sdFVs), anti-idiotypic (anti-Id)
antibodies, epitope-binding fragments of such antibodies, and the
like.
[0042] The term "monoclonal antibody" refers to an antibody to a
single determinant of an antigen and is an identical antibody,
which is obtained from a population of substantially homogeneous
antibodies, that is, an identical antibody constituting the
population, excluding possible naturally occurring mutations that
may be present in trivial amounts.
[0043] The term "epitope" refers to a protein determinant to which
an antibody is capable of specifically binding. Epitopes usually
consist of a group of chemically active surface molecules, such as
amino acid or sugar side chains, and generally have not only
specific three-dimensional structural characteristics but also
specific charge characteristics. Three-dimensional epitopes are
distinguished from non-three-dimensional epitopes in that a bond to
the former is broken in the presence of a denatured solvent, while
a bond to the latter is not broken.
[0044] The term "complete antibody" refers to a structure having
two full-length light chains and two full-length heavy chains,
wherein each light-chain is linked to a corresponding heavy-chain
by a disulfide bond. The heavy-chain constant region has gamma
(.gamma.), mu (.mu.), alpha (.alpha.), delta (.delta.) and epsilon
(.epsilon.) types, and is subclassified into gamma 1 (.gamma.1),
gamma 2 (.gamma.2), gamma 3 (.gamma.3), gamma 4 (.gamma.4), alpha 1
(.alpha.1) and alpha 2 (.alpha.2). The light-chain constant region
has kappa (.kappa.) and lambda (.lamda.) types.
[0045] The antigen-binding fragment of an antibody or antibody
fragment is a fragment that has antigen-binding capability, and
includes Fab, F(ab'), F(ab')2, Fv and the like. Among the antibody
fragments, Fab refers to a structure including a variable region of
each of the heavy chain and the light chain, the constant region of
the light chain, and the first constant domain (CH1) of the heavy
chain, each having one antigen-binding site. Fab' is different from
Fab in that it further includes a hinge region including at least
one cysteine residue at the C-terminus of the CH1 domain of the
heavy chain. F(ab')2 is created by a disulfide bond between
cysteine residues in the hinge region of Fab'. Fv is the minimal
antibody fragment having only a heavy-chain variable region and a
light-chain variable region. Two-chain Fv is a fragment in which
the variable region of the heavy chain and the variable region of
the light chain are linked by a non-covalent bond, and single-chain
Fv (scFv) is a fragment wherein the variable region of the heavy
chain and the variable region of the light chain are generally
linked by a covalent bond via a peptide linker therebetween, or are
directly linked at the C-terminus, forming a dimer-shaped
structure, like the two-chain Fv. Such antibody fragments may be
obtained using proteases (e.g. Fab can be obtained by
restriction-cleaving the complete antibody with papain, and the
F(ab')2 fragment can be obtained by cleaving the complete antibody
with pepsin), and may be also prepared using genetic recombination
techniques.
[0046] The "Fv" fragment is an antibody fragment comprising
complete antibody recognition and binding sites. Such a region
includes a dimer that consists of one heavy-chain variable domain
and one light-chain variable domain substantially tightly
covalently linked to each other, for example, through scFv.
[0047] A "Fab" fragment comprises a variable domain and a constant
domain of the light-chain and a variable domain and a first
constant domain (CH1) of the heavy chain. A F(ab')2 antibody
fragment generally includes a pair of Fab fragments covalently
linked near the carboxyl terminal thereof via a hinge cysteine
therebetween.
[0048] The "single-chain Fv" or "scFv" antibody fragment includes
VH and VL domains of the antibody, wherein these domains are
present in a single polypeptide chain. The Fv polypeptide may
further include a polypeptide linker between the VH domain and the
VL domain in order for the scFv to form a desired structure for
antigen binding.
[0049] In one embodiment, the antibody of the present invention is
in an Fv form (for example, scFv) or a complete antibody form. In
addition, the heavy-chain constant region may be selected from
gamma (.gamma.), mu (u), alpha (.alpha.), delta (.delta.) and
epsilon (c) isotypes. For example, the constant region may be gamma
1 (IgG1), gamma 3 (IgG3), or gamma 4 (IgG4). The light-chain
constant region may be kappa or lambda.
[0050] As used herein, the term "heavy chain" encompasses both a
full-length heavy chain, which includes a variable domain (VH),
comprising an amino acid sequence having a variable region sequence
sufficient for imparting specificity to an antigen and three
constant domains (CH1, CH2 and CH3), and a fragment thereof. As
used herein, the term "light chain" encompasses both a full-length
light chain, which includes a variable domain (VL) comprising an
amino acid sequence having a variable region sequence sufficient
for imparting specificity to an antigen and a constant domain (CL),
and a fragment thereof.
[0051] A part of the heavy chain and/or light chain is identical to
or homologous with the corresponding sequence in an antibody
derived from a particular species or belonging to a particular
antibody class or subclass, while the remainder of the chain(s)
includes "chimeric" antibodies (immunoglobulins) which are
identical to or homologous with corresponding sequences in an
antibody derived from another species or belonging to another
antibody class or subclass, as well as fragments of such antibody
exhibiting the desired biological activity.
[0052] As used herein, the term "antibody variable domain" refers
to the light- and heavy-chain regions of an antibody molecule
comprising the amino acid sequences of a
complementarity-determining region (CDR; i.e., CDR1, CDR2, and
CDR3) and a framework region (FR). VH refers to a variable domain
of the heavy chain. VL refers to a variable domain of the light
chain.
[0053] The term "complementarity-determining region" (CDR; that is,
CDR1, CDR2, and CDR3), refers to an amino acid residue of the
antibody variable domain, which is necessary for antigen binding.
Each variable domain typically has three CDR regions, identified as
CDR1, CDR2, and CDR3. In one embodiment, the
complementarity-determining region comprises a heavy-chain CDR1
represented by SEQ ID NO: 3, 6, 9 or 12, a heavy-chain CDR2
represented by SEQ ID NO: 4, 7 or 10, a heavy-chain CDR3
represented by SEQ ID NO: 5, 8, 11 or 13, a light-chain CDR1
represented by SEQ ID NO: 14, 17, 20, 23 or 25, a light-chain CDR2
represented by SEQ ID NO: 15, 18, 21 or 26, and a light-chain CDR3
represented by SEQ ID NO: 16, 19, 22, 24 or 27.
[0054] In the present invention, the antibody binding to TIGIT or
antigen-binding fragment thereof may comprise a heavy-chain CDR1 of
SEQ ID NO: 3, a heavy-chain CDR2 of SEQ ID NO: 4, a heavy-chain
CDR3 of SEQ ID NO: 5, a light-chain CDR1 of SEQ ID NO: 14, a
light-chain CDR2 of SEQ ID NO: 15, and a light-chain CDR3 of SEQ ID
NO: 16;
[0055] a heavy-chain CDR1 of SEQ ID NO: 6, a heavy-chain CDR2 of
SEQ ID NO: 7, a heavy-chain CDR3 of SEQ ID NO: 8, a light-chain
CDR1 of SEQ ID NO: 17, a light-chain CDR2 of SEQ ID NO: 18, and a
light-chain CDR3 of SEQ ID NO: 19;
[0056] a heavy-chain CDR1 of SEQ ID NO: 9, a heavy-chain CDR2 of
SEQ ID NO: 10, a heavy-chain CDR3 of SEQ ID NO: 11, a light-chain
CDR1 of SEQ ID NO: 20, a light-chain CDR2 of SEQ ID NO:21, and a
light-chain CDR3 of SEQ ID NO: 22;
[0057] a heavy-chain CDR1 of SEQ ID NO: 9, a heavy-chain CDR2 of
SEQ ID NO: 10, a heavy-chain CDR3 of SEQ ID NO: 11, a light-chain
CDR1 of SEQ ID NO: 23, a light-chain CDR2 of SEQ ID NO: 21, and a
light-chain CDR3 of SEQ ID NO: 24; or
[0058] a heavy-chain CDR1 of SEQ ID NO: 12, a heavy-chain CDR2 of
SEQ ID NO: 10, a heavy-chain CDR3 of SEQ ID NO: 13, a light-chain
CDR1 of SEQ ID NO: 25, a light-chain CDR2 of SEQ ID NO: 26, and a
light-chain CDR3 of SEQ ID NO: 27.
[0059] The term "framework region" (FR) refers to a variable domain
residue other than a CDR residue. Each variable domain typically
has four FRs, identified as FR1, FR2, FR3, and FR4.
[0060] The TIGIT antibody may have a monovalent or divalent form,
and may comprise single or double chains. Functionally, the binding
affinity of the TIGIT antibody ranges from 10.sup.-5 M to
10.sup.-12 M. For example, the binding affinity of the TIGIT
antibody may range from 10.sup.-6 M to 10.sup.-12 M, 10.sup.-7 M to
10.sup.-12 M, 10.sup.-8 M to 10.sup.-12 M, 10.sup.-9 M to
10.sup.-12 M, 10.sup.-5 M to 10.sup.-11 M, 10.sup.-6 M to
10.sup.-11 M, 10.sup.-7 M to 10.sup.-11 M, 10.sup.-8 M to
10.sup.-11 M, 10.sup.-9 M to 10.sup.-11 M, 10.sup.-10 M to
10.sup.-11 M, 10.sup.-3 M to 10.sup.-10 M, 10.sup.-6 M to
10.sup.-10 M, 10.sup.-7 M to 10.sup.-10 M, 10.sup.-8 M to
10.sup.-10 M, 10.sup.-9 M to 10.sup.-10 M, 10.sup.-5 M to 10.sup.-9
M, 10.sup.-6 M to 10.sup.-9 M, 10.sup.-7 M to 10.sup.-9 M,
10.sup.-8 M to 10.sup.-9 M, 10.sup.-5 M to 10.sup.-8 M, 10.sup.-6 M
to 10.sup.-8 M, 10.sup.-7 M to 10.sup.-8 M, 10.sup.-5 M to
10.sup.-7 M, 10.sup.-6 M to 10.sup.-7 M, or 10.sup.-5 M to
10.sup.-6 M.
[0061] The antibody binding to TIGIT or an antigen-binding fragment
thereof may comprise at least one heavy-chain variable region
selected from the group consisting of SEQ ID NOS: 28 to 32. In
addition, the antibody binding to TIGIT or an antigen-binding
fragment thereof may comprise at least one light-chain variable
region selected from the group consisting of SEQ ID NOS: 34 to
38.
[0062] Specifically, the antibody binding to TIGIT or an
antigen-binding fragment thereof comprises: the heavy-chain
variable region of SEQ ID NO: 28 and the light-chain variable
region of SEQ ID NO: 34; the heavy-chain variable region of SEQ ID
NO: 29 and the light-chain variable region of SEQ ID NO: 35; the
heavy-chain variable region of SEQ ID NO: 30 and the light-chain
variable region of SEQ ID NO: 36; the heavy-chain variable region
of SEQ ID NO: 31 and the light-chain variable region of SEQ ID NO:
37; or the heavy-chain variable region of SEQ ID NO: 32 and the
light-chain variable region of SEQ ID NO: 38.
[0063] "Phage display" is a technique for displaying a mutant
polypeptide as a fusion protein with at least a part of a coat
protein on the surface of the particle of a phage, for example a
fibrous phage. The usefulness of phage display is to rapidly and
efficiently classify sequences that bind to target antigens with
high affinity in large libraries of randomized protein mutants.
Displaying peptides and protein libraries on phages has been used
to screen millions of polypeptides in order to identify
polypeptides with specific binding properties.
[0064] Phage display technology has offered a powerful tool for
producing and screening novel proteins that bind to specific
ligands (e.g., antigens). Using phage display technology, large
libraries of protein mutants can be generated, and sequences
binding with high affinity to target antigens can be rapidly
classified. The nucleic acid encoding mutant polypeptides is fused
with a nucleic acid sequence encoding a viral coat protein, e.g. a
gene III or gene VIII protein. A monophasic phage display system,
in which a nucleic acid sequence encoding a protein or polypeptide
is fused with a nucleic acid sequence encoding a part of a gene III
protein, has been developed. In the monophasic display system, a
fused gene is expressed at a low level, and a wild-type gene III
protein is also expressed, and thus particle infectivity is
maintained.
[0065] It is important to demonstrate the expression of peptides on
the fibrous phage surface and the expression of functional antibody
fragments in the peripheral cytoplasm of E. coli for the
development of antibody phage display libraries. Libraries of
antibody- or antigen-binding polypeptides are produced through a
number of methods, for example, methods of modifying a single gene
by inserting a random DNA sequence or cloning a related gene
sequence. Libraries can be screened regarding the expression of
antibody- or antigen-binding proteins having desired
characteristics.
[0066] Phage display technology has several advantages over
conventional hybridomas and recombinant methods for producing
antibodies having desired characteristics. This technique provides
the generation of large antibody libraries with a variety of
sequences within a short time without using animals. The production
of hybridomas and the production of humanized antibodies may
require a production time of several months. In addition, since no
immunity is required, the phage antibody libraries can generate
antibodies against antigens that are toxic or have low
antigenicity. The phage antibody libraries can also be used to
produce and identify novel therapeutic antibodies.
[0067] Techniques for generating human antibodies from immunized or
non-immunized human germline sequences or naive B cell Ig
repertoires using phage display libraries can be used. Naive or
non-immunogenic antigen-binding libraries can be produced using
various lymphatic tissues.
[0068] Techniques for identifying and separating high-affinity
antibodies from phage display libraries are important for the
separation of new therapeutic antibodies. The separation of
high-affinity antibodies from libraries depends on the size of the
libraries, the production efficiency in bacterial cells, and the
variety of libraries. The size of the libraries is reduced by
improper folding of the antibody- or antigen-binding protein and
inefficient production due to the presence of the stop codon.
Expression in bacterial cells may be inhibited by improper folding
of the antibody- or antigen-binding domain. The expression can be
improved by alternately mutating residues on the surface of the
variable/constant interfaces or the selected CDR residues. The
sequence of the framework region is an element that enables proper
folding when generating antibody phage libraries in bacterial
cells.
[0069] It is important to generate various libraries of antibody-
or antigen-binding proteins in the separation of high-affinity
antibodies. CDR3 regions have often been found to participate in
antigen binding. Since the CDR3 region in the heavy chain varies
considerably with regard to size, sequence and
structural/dimensional morphology, various libraries can be
produced using the same.
[0070] Also, diversity can be created by randomizing the CDR
regions of variable heavy and light chains using all 20 amino acids
at each position. The use of all 20 amino acids results in the
production of antibody sequences having increased diversity and an
increased chance of identifying new antibodies.
[0071] The antibody or antibody fragment of the present invention
may comprise the sequence of the anti-TIGIT antibody mentioned
herein as well as biological equivalents thereto, as long as it can
specifically recognize TIGIT. For example, additional variations
can be made to the amino acid sequence of the antibody in order to
further improve the binding affinity and/or other biological
properties of the antibody. Such variations include, for example,
deletion, insertion and/or substitution of the amino acid sequence
residues of the antibody. Such amino acid variations are based on
the relative similarity of amino acid side chain substituents, such
as the hydrophobicity, hydrophilicity, charge, and size thereof. It
can be seen through analysis of the size, shape and type of amino
acid side chain substituents that all of arginine, lysine, and
histidine are positively charged residues; alanine, glycine, and
serine have similar sizes; and phenylalanine, tryptophan, and
tyrosine have similar structure. Thus, based on these
considerations, arginine, lysine, and histidine; alanine, glycine,
and serine; and phenylalanine, tryptophan, and tyrosine are
considered to be biologically functional equivalents.
[0072] Taking into consideration variations having biologically
equivalent activity, the antibody or a nucleotide molecule encoding
the same according to the present invention is interpreted to
comprise a sequence having substantial identity with the sequence
set forth in the sequence number. The term "substantial identity"
means that a sequence has a homology of at least 90%, most
preferably a homology of at least 95%, at least 96%, at least 97%,
at least 98%, or at least 99%, when aligning the sequence of the
present invention with any other sequence so as to correspond to
each other as closely as possible and analyzing the aligned
sequence using algorithms commonly used in the art. Alignment
methods for sequence comparison are well-known in the art. The NCBI
Basic Local Alignment Search Tool (BLAST) is accessible through
NCBI or the like, and can be used in conjunction with sequence
analysis programs such as blastp, blastm, blastx, tblastn and
tblastx over the Internet. BLAST is available at
www.ncbi.nlm.nih.gov/BLAST/. A method of comparing sequence
homology using this program can be found at
www.ncbi.nlm.nih.gov/BLAST/blast help.html.
[0073] Based on this, the antibody or antigen-binding fragment
thereof according to the present invention can have homology of
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more with the
sequence disclosed herein or the entirety thereof. Homology can be
determined through sequence comparison and/or alignment by methods
known in the art. For example, the percentage sequence homology of
the nucleic acid or protein according to the present invention can
be determined using a sequence comparison algorithm (i.e., BLAST or
BLAST 2.0), manual alignment, or visual inspection.
[0074] In another aspect, the present invention is directed to a
nucleic acid encoding the antibody or an antigen-binding fragment
thereof.
[0075] By isolating the nucleic acid encoding the antibody or
antigen-binding fragment thereof according to the present
invention, an antibody or antigen-binding fragment thereof can be
produced in a recombinant manner. The nucleic acid is isolated and
inserted into a replicable vector, followed by further cloning
(amplification of DNA) or further expression. Based thereon, in
another aspect, the present invention is directed to a vector
comprising the nucleic acid.
[0076] The term "nucleic acid" is intended to encompass both DNA
(gDNA and cDNA) and RNA molecules, and a nucleotide, which is the
basic constituent unit of nucleic acids, includes naturally derived
nucleotides as well as analogues thereof, in which sugar or base
moieties are modified. The sequence of the nucleic acid encoding
heavy- and light-chain variable regions of the present invention
can vary. Such variation includes addition, deletion, or
non-conservative or conservative substitution of nucleotides.
[0077] The DNA encoding the antibody can be easily separated or
synthesized using conventional procedures (for example, using an
oligonucleotide probe capable of specifically binding to DNA
encoding heavy and light chains of the antibody). A variety of
vectors are obtainable. Vector components generally include, but
are not limited to, one or more of the following components: signal
sequences, replication origins, one or more marker genes, enhancer
elements, promoters, and transcription termination sequences.
[0078] As used herein, the term "vector" refers to a means for
expressing target genes in host cells, and includes plasmid
vectors, cosmid vectors, and viral vectors such as bacteriophage
vectors, adenovirus vectors, retroviral vectors, and
adeno-associated viral vectors. The nucleic acid encoding the
antibody in the vector is operably linked to a promoter.
[0079] The term "operably linked" means functional linkage between
a nucleic acid expression regulation sequence (e.g., an array of
the binding site of the promoter, signal sequence, or transcription
regulator) and another nucleic acid sequence, and enables the
regulation sequence to regulate transcription and/or translation of
the other nucleic acid sequence.
[0080] When a prokaryotic cell is used as a host, it generally
includes a potent promoter capable of conducting transcription
(such as a tac promoter, lac promoter, lacUV5 promoter, lpp
promoter, pLA promoter, pRA promoter, racy promoter, amp promoter,
recA promoter, SP6 promoter, trp promoter, or T7 promoter), a
ribosome-binding site for initiation of translation, and a
transcription/translation termination sequence. In addition, for
example, when a eukaryotic cell is used as a host, it includes a
promoter derived from the genome of a mammalian cell (e.g., a
metallothionein promoter, a .beta.-actin promoter, a human
hemoglobin promoter or a human muscle creatine promoter), or a
promoter derived from a mammalian virus (e.g., an adenovirus late
promoter, vaccinia virus 7.5 K promoter, SV40 promoter,
cytomegalovirus (CMV) promoter, HSV tk promoter, mouse mammary
tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus
promoter, Epstein-Barr virus (EBV) promoter, or Rous sarcoma virus
(RSV) promoter), and generally has a polyadenylation sequence as a
transcription termination sequence.
[0081] Optionally, the vector may be fused with another sequence in
order to facilitate purification of the antibody expressed thereby.
The sequence to be fused therewith may include, for example,
glutathione S-transferase (Pharmacia, USA), maltose-binding protein
(NEB, USA), FLAG (IBI, USA), 6.times.His (hexahistidine; Qiagen,
USA) and the like.
[0082] The vector comprises antibiotic resistance genes commonly
used in the art as selectable markers, and examples thereof include
genes conferring resistance to ampicillin, gentamycin,
carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin,
neomycin, and tetracycline.
[0083] In another aspect, the present invention is directed to a
cell transformed with the above-mentioned vector. The cell used to
produce the antibody of the present invention may be a prokaryote,
yeast, or higher eukaryotic cell, but is not limited thereto.
[0084] Prokaryotic host cells such as Escherichia coli, strains of
the genus Bacillus, such as Bacillus subtilis and Bacillus
thuringiensis, Streptomyces spp., Pseudomonas spp. (for example,
Pseudomonas putida), Proteus mirabilis and Staphylococcus spp. (for
example, Staphylococcus carnosus) may be used.
[0085] Interest in animal cells is the greatest, and examples of
useful host cell lines include, but are not limited to, COS-7, BHK,
CHO, CHOK1, DXB-11, DG-44, CHO/-DHFR, CV1, COS-7, HEK293, BHK, TM4,
VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5,
FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2/0, NS-0, U20S, and
HT1080.
[0086] In another aspect, the present invention is directed to a
method of producing the antibody or antigen-binding fragment
thereof comprising (a) culturing the cell; and (b) recovering an
antibody or antigen-binding fragment thereof from the cultured
cell.
[0087] The cells can be cultured in various media. Any commercially
available medium can be used as a culture medium without
limitation. All other essential supplements well-known to those
skilled in the art may be included in appropriate concentrations.
Culture conditions such as temperature and pH are those that are
conventionally used with host cells selected for expression, as
will be apparent to those skilled in the art.
[0088] The recovery of the antibody or antigen-binding fragment
thereof can be carried out, for example, by centrifugation or
ultrafiltration to remove impurities from and purify the resulting
product using, for example, affinity chromatography. Other
additional purification techniques, such as anion or cation
exchange chromatography, hydrophobic interaction chromatography,
and hydroxyapatite (HA) chromatography, may be used.
[0089] In another aspect, the present invention is directed to a
composition for preventing or treating cancer or tumors comprising
the antibody as an active ingredient.
[0090] For example, the present invention is directed to a
pharmaceutical composition for preventing or treating cancer or
tumors comprising (a) a pharmaceutically effective amount of the
antibody to TIGIT or antigen-binding fragment thereof according to
the present invention; and (b) a pharmaceutically acceptable
carrier. In another aspect, the present invention is directed to a
method for preventing or treating cancer or tumors comprising
administering the antibody to TIGIT or an antigen-binding fragment
thereof according to the present invention to a patient in an
effective amount determined according to the requirements of the
patent.
[0091] In the present invention, the terms "cancer" or "tumor"
typically refer to or mean the physiological condition of a mammal
characterized by uncontrolled cell growth/proliferation.
[0092] The composition uses the anti-TIGIT antibody or
antigen-binding fragment thereof according to the present invention
described above as an active ingredient, and thus redundant
descriptions will be omitted.
[0093] As demonstrated in Examples given below, the antibody or
antigen-binding fragment thereof according to the present invention
binds to TIGIT with high affinity, and can be useful for treating
cancer that evades anti-tumor T-cell activity.
[0094] The cancer or carcinoma that can be treated with the
composition of the present invention is not particularly limited,
and includes both solid cancer and blood cancer. Examples of such
cancer include, but are not limited to, lymphoma, leukemia, and
multiple myeloma.
[0095] The specific example according to the present invention
demonstrated that TIGIT is expressed to a high level in regulatory
T cells of multiple myeloma patients, and treatment with the
antibody or antigen-binding fragment thereof according to the
present invention resulted in a significant increase in IFN-.gamma.
secretion upon anti-CD3/anti-CD28 stimulation to PBMC of multiple
myeloma patients.
[0096] For example, the cancer may be selected from the group
consisting of skin cancer such as melanoma, liver cancer,
hepatocellular carcinoma, gastric cancer, breast cancer, lung
cancer, ovarian cancer, bronchial cancer, nasopharyngeal cancer,
laryngeal cancer, pancreatic cancer, bladder cancer, colorectal
cancer, colon cancer, uterine cervical cancer, brain cancer,
prostate cancer, bone cancer, thyroid cancer, parathyroid cancer,
renal cancer, esophageal cancer, biliary tract cancer, testicular
cancer, rectal cancer, head and neck cancer, cervical cancer,
ureteral cancer, osteosarcoma, neurocytoma, fibrosarcoma,
rhabdomyosarcoma, astrocytoma, neuroblastoma, and neuroglioma, but
is not limited thereto.
[0097] The specific example according to the present invention
demonstrated that when a human colorectal cancer cell xenograft
model was treated with the antibody or antigen-binding fragment
thereof according to the present invention, an excellent effect of
inhibiting tumor growth was obtained.
[0098] In another aspect, the present invention is directed to a
composition for preventing or treating cancer by using the antibody
or antigen-binding fragment thereof in combination with another
anticancer therapy or administering the same in combination with
another drug, for example, an anti-cancer agent.
[0099] For example, the present invention is directed to a
composition for co-administration for preventing or treating cancer
or tumors comprising (a) a pharmaceutically effective amount of the
antibody to TIGIT or antigen-binding fragment thereof according to
the present invention; and (b) a pharmaceutically acceptable
carrier. In another aspect, the present invention is directed to a
co-administration method for preventing or treating cancer or
tumors comprising administering the antibody to TIGIT or
antigen-binding fragment thereof according to the present invention
to a patient in an effective amount as required by the patent.
[0100] The composition uses the anti-TIGIT antibody or
antigen-binding fragment thereof according to the present invention
described above as an active ingredient, and thus redundant
descriptions will be omitted.
[0101] By administering other anticancer agents in addition to the
antibody in combination therewith, it is possible to effectively
target tumor cells overexpressing TIGIT and to increase anti-tumor
T-cell activity and to thereby enhance the immune response
targeting tumor cells.
[0102] The antibody may be used in combination with: other
anti-neoplastic or immunogenic agents [e.g., attenuated cancer
cells, tumor antigens (including recombinant proteins, peptides,
and carbohydrate molecules), antigen transfer cells, for example,
tumor-derived antigens or nucleic-acid-pulsed dendritic cells,
immunostimulating cytokines (e.g., IL-2, IFN.alpha.2, and GM-CSF),
and cells transfected with genes encoding immunostimulating
cytokines (including for example but not limited to GM-CSF)];
standard cancer therapy (e.g. chemotherapy, radiotherapy or
surgery); or other antibodies (including but not limited to
antibodies other than TIGIT antibodies, VEGF, EGFR, Her2/neu, VEGF
receptors, other growth factor receptors, CD20, CD40, CTLA-4,
OX-40, 4-IBB, and ICOS).
[0103] The drug, for example, an anticancer agent, may be an immune
checkpoint inhibitor, but is not limited thereto.
[0104] In another aspect, the present invention is directed to a
composition for preventing or treating cancer using the antibody or
antigen-binding fragment thereof in combination with an immune
checkpoint inhibitor.
[0105] In the present invention, the immune checkpoint inhibitor
refers to an agent that is capable of inducing T-cell activation by
blocking movement of the T-cell inhibitory signal to the site where
antigen-presenting cells (APCs) meet with immune cells, for
example, T cells. The immune checkpoint inhibitor may be, for
example, a drug targeting PD-1, PD-L1, or CTLA-4, but is not
limited thereto.
[0106] The immune checkpoint inhibitor may specifically be an
anti-CTLA-4 antibody, an anti-PD-1 antibody, or an anti-PD-L1
antibody, but is not limited thereto. Specifically, the immune
checkpoint inhibitor may be ipilimumab, nivolumab, pembrolizumab,
atezolizumab, avelumab, durvalumab or the like, but is not limited
thereto.
[0107] A specific example of the present invention showed that when
the anti-TIGIT antibody or antigen-binding fragment thereof is used
in combination with the anti-PD-1 antibody, pembrolizumab, the T
cell reactivity can be increased, T-cell proliferation can be
increased, and IFN-.gamma. secretion can be increased, compared to
when the anti-TIGIT antibody is used alone.
[0108] The term "used in combination", "combined use" or
"co-administration" means that the anti-TIGIT antibody or
antigen-binding fragment thereof and another drug, for example, an
anticancer agent, can be administered simultaneously, sequentially,
or in reverse order. The composition of the present invention may
be administered as a single therapeutic agent or in combination
with other therapeutic agents, and may be administered sequentially
or simultaneously with a conventional therapeutic agent.
[0109] The cancer, which is the disease to which the composition
can be applied, includes typical cancers that respond to
immunotherapy, as well as cancers that have not been related with
immunotherapy to date. Non-limiting examples of cancer that is the
target of treatment include melanoma (e.g., metastatic malignant
melanoma), kidney cancer (e.g., clear cell carcinoma), prostate
cancer (e.g., hormone refractory prostate adenocarcinoma),
pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer
(e.g. non-small cell lung cancer), esophageal cancer, head and neck
squamous cell carcinoma, liver cancer, ovarian cancer, cervical
cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma,
blood cancers such as multiple myeloma, and other neoplastic
carcinomas. In addition, the cancer according to the present
invention includes refractory or recurrent cancer, the growth of
which can be inhibited using the antibody of the present
invention.
[0110] Antibodies or antibody fragments can be used alone or in
combination with vaccines to stimulate an immune response against
pathogens, toxins, and self-antigens. Antibodies or antigen-binding
fragments thereof can be used to stimulate an immune response
against viruses that infect humans, and examples of such viruses
include, but are not limited to, human immunodeficiency virus,
hepatitis virus classes A, B and C, Epstein-Barr virus, human
cytomegalovirus, human papilloma virus, and herpes viruses.
Antibodies or antigen-binding fragments thereof can be used to
stimulate an immune response to infection with bacterial or fungal
parasites and other pathogens.
[0111] The pharmaceutically acceptable carrier comprised in the
composition according to the present invention may be commonly used
in preparations, and may include, but is not limited to, lactose,
dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium
phosphate, alginate, gelatin, calcium silicate, microcrystalline
cellulose, polyvinylpyrrolidone, water, syrup, methylcellulose,
methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium
stearate, mineral oil, and the like. The composition according to
the present invention may further comprise a lubricant, a wetting
agent, a sweetener, a flavoring agent, an emulsifier, a suspension
agent, a preservative, or the like, in addition to the ingredients
described above.
[0112] The pharmaceutical composition according to the present
invention may be administered orally or parenterally. The
parenteral administration may be intravenous injection,
subcutaneous injection, intramuscular injection, intraperitoneal
injection, endothelial administration, topical administration,
intranasal administration, pulmonary administration, rectal
administration, or the like.
[0113] Upon oral administration, since proteins or peptides are
digested, an oral composition should be coated with an active drug
or formulated so as to protect the same from degradation in the
stomach. In addition, the pharmaceutical composition may be
administered using any device capable of delivering the active
substance to target cells.
[0114] The suitable dose of the pharmaceutical composition
according to the present invention may vary depending on factors
such as the formulation method, administration method, and age,
body weight, gender, pathological conditions, diet, administration
time, administration route, excretion rate, and responsiveness of
the patient, and a general physician of ordinary skill can easily
determine and prescribe a dose effective for the desired treatment
or prevention. For example, the daily dose of the pharmaceutical
composition according to the present invention may be within the
range of 0.0001 to 100 mg/kg. The term "pharmaceutically effective
amount" as used herein may mean an amount sufficient to prevent or
treat cancer.
[0115] The pharmaceutical composition according to the present
invention may be prepared into a unit dose form, or may be
incorporated into a multi-dose container through formulation using
a pharmaceutically acceptable carrier and/or excipient according to
a method that can be easily implemented by those skilled in the art
to which the present invention pertains. Here, the formulation may
be in the form of a solution, a suspension or an emulsion in an oil
or aqueous medium, or may be in the form of an extract, a powder, a
suppository, a granule, a tablet, or a capsule. The composition may
further comprise a dispersant or a stabilizer.
EXAMPLE
[0116] Hereinafter, the present invention will be described in more
detail with reference to examples. However, it will be obvious to
those skilled in the art that these examples are provided only for
illustration of the present invention, and should not be construed
as limiting the scope of the present invention.
Example 1. Screening of TIGIT Antibodies
[0117] Human synthetic library phage was reacted in a tube coated
with human TIGIT-His antigen for 2 hours. After the reaction, the
reaction product was washed 4 times with washing buffer (PBS+0.05%
Tween 20) and reacted with elution buffer (1% BSA/0.1M glycine, pH
2.0) at room temperature for 10 minutes, and then the phage was
recovered. XLI-Blue competent cells were infected with the
recovered phage and incubated at 37.degree. C. for 1 hour. Then,
the result was treated with 1 mL of a VCS M13 helper phage,
incubated at 37.degree. C. for 1 hour, further treated with 80 mL
of SB medium, 100 .mu.l of kanamycin, and 100 .mu.l of
carbenicillin, and then incubated at 37.degree. C. for 20 hours.
After incubation, the supernatant was recovered, the phage was
precipitated with a PEG solution (20% PEG, 15% NaCl), re-suspension
was performed with 2 ml of 1% BSA/PBS, and the result was used for
the next panning.
[0118] The human antibody library was displayed on the phage
surface to screen human TIGIT-specific human antibodies and then
the concentration of the antigen, TIGIT, was changed, as the number
of rounds increased, and panning was performed for a total of 3
rounds. As the number of times panning was performed increased, the
concentration of TIGIT was decreased from 100 nM to 1 nM. The
output titer of the phage is shown in Table 1.
TABLE-US-00002 TABLE 1 Phage output titer Round Antigen
Concentration (nM) Output titer 1 TIGIT-HIS 100 nM 5.0E+10.sup.7 2
TIGIT-HIS 50 nM 3.2E+10.sup.6 3 TIGIT-HIS 1 nM 7.8E+10.sup.5
Example 2. Determination of Binding Capacity to TIGIT
[0119] 2.1 Determination of Binding Capacity of ScFv-Type
Anti-TIGIT Antibody
[0120] 50 .mu.l of a human TIGIT-FC antigen was added at a
concentration of 3 .mu.g/ml to an ELISA plate, incubated at
4.degree. C. overnight, and then blocked with 1% BSA in PBS at room
temperature for 2 hours. A total of 16 scFv anti-TIGIT antibodies
were serially diluted by 1/3 in PBS at a starting concentration of
80 nM, fed in an amount of 50 .mu.l into a human TIGIT-Fc plate,
and then allowed to react at room temperature for 2 hours. The
result was washed 3 times with PBST (0.05% Tween 20 in PBS) and was
reacted with 50 .mu.l of anti-His-HRP diluted 1/1000 in PBS at room
temperature for 1 hour. The result was washed 3 times with PBST
(0.05% Tween 20 in PBS), and 50 .mu.l of TMB solution was added
thereto. The result was reacted at room temperature for 5 minutes,
50 .mu.l of a TMB stop solution was added thereto, and the
absorbance was measured at 450 nm with an ELISA leader.
[0121] The results are shown in FIG. 1. A total of 11 clones (1C3,
1E4, 1E12, 1F2, 1F10, 1G8, WIN_1B6, WIN_1B11, WIN_1D2, 1E8, and
1B11) were selected based on the EC50 value.
[0122] 2.2 Determination of Binding Affinity of IgG-Type Anti-TIGIT
Antibody
[0123] Among the scFv anti-human TIGIT antibodies, 11 types of
antibodies having high binding affinity to human TIGIT were
converted to IgG antibodies. 50 .mu.l of the human TIGIT-Fc antigen
was added at a concentration of 3 .mu.g/ml to an ELISA plate,
incubated at 4.degree. C. overnight, and then blocked with 1% BSA
in PBS at room temperature for 2 hours. The total of 11 IgG-type
anti-human TIGIT antibodies were serially diluted by 1/2 in PBS at
a starting concentration of 6.6 nM, and 50 .mu.l thereof was added
to a human TIGIT-Fc plate and then allowed to react at room
temperature for 2 hours. The result was washed 3 times with PBST
(0.05% Tween 20 in PBS), and 50 .mu.l of anti-His-HRP was diluted
1/1000 in PBS and then reacted at room temperature for 1 hour. The
result was washed 3 times with PBST (0.05% Tween 20 in PBS), and 50
.mu.l of a TMB solution was added thereto. The result was allowed
to react at room temperature for 5 minutes, 50 .mu.l of a TMB stop
solution was added thereto, and the absorbance was measured at 450
nm in an ELISA leader. The results are shown in Table 2 and FIG.
2.
TABLE-US-00003 TABLE 2 Clone 1B11 1C3 1E4 1E12 1F2 1F10 1G8 WIN-1B6
WIN-1B11 WIN-1D2 1E8 EC50 (nM) 4.21 3.56 1.49 2.94 1.78 5.35 1.63
0.86 1.63 1.75 1.73
[0124] It was found that 1E4, 1F2, 1G8, WIN-1D2, and 1E8 had a
binding affinity of about 1 nM EC50.
Example 3. Determination of Competitiveness of TIGIT Candidate
Antibody for PVR, Ligand of TIGIT
[0125] 3.1 Human PVR Expression and Purification
[0126] The amino acid (SEQ ID NO: 2) of the ECD (extracellular
domain) Gly27-Asn343 region of the human PVR sequence provided by
GenBank AAH15542.1 was synthesized.
TABLE-US-00004 Human PVR (Gly27-Asn343) SEQ ID NO: 2
GDVVVQAPTQVPGFLGDSVTLPCYLQVPNMEVTHVSQLTWARHGESGSMA
VFHQTQGPSYSESKRLEFVAARLGAELRNASLRMFGLRVEDEGNYTCLFV
TFPQGSRSVDIWLRVLAKPQNTAEVQKVQLTGEPVPMARCVSTGGRPPAQ
ITWHSDLGGMPNTSQVPGFLSGTVTVTSLWILVPSSQVDGKNVTCKVEHE
SFEKPQLLTVNLTVYYPPEVSISGYDNNWYLGQNEATLTCDARSNPEPTG
YNWSTTMGPLPPFAVAQGAQLLIRPVDKPINTTLICNVTNALGARQAELT
VQVKEGPPSEHSGMSRN
[0127] PCR was performed with hPVR-F (5'
GGCCCAGGCGGCCGGCGA-3')/hPVR-R (5'-GGCCAGGCTGGCCGTTCC-3') primers,
using the synthesized human PVR as a template, and then inserted
into the pclw-huIgG4 vector using a Sfi I enzyme. 30 .mu.g of
light-chain DNA and heavy-chain DNA at a ratio of 1:1 was
transiently expressed in 30 id of 2.5.times.10.sup.6 Expi293F.TM.
cells/ml using an ExpiFectamine 293 reagent and then incubated for
10 days. The cell culture solution (supernatant) was filtered using
a 0.22 .mu.m bottle top filter, and was then reacted with 200 .mu.l
of MabSelect Xtra beads while mixing in a bio-rotator for 2 hours.
After incubation, the bead and antibody mixture was added to the
protein A column. The protein A column was washed twice with 2 ml
of binding buffer. A total of five elution fractions were collected
using 200 .mu.l of protein A elution buffer in the protein A
column. The eluted antibodies were desalted by centrifugation at
1,000 rpm for 5 minutes using a Zeba spin desalting column.
[0128] 3.2 Determination of Competitiveness
[0129] 50 .mu.l of a human TIGIT-Fc antigen was added at a
concentration of 3 .mu.g/ml to an ELISA plate, incubated at
4.degree. C. overnight, and blocked with 1% BSA in PBS at room
temperature for 2 hours. Biotinylated human PVR as a ligand and the
anti-human TIGIT antibody were mixed at various molar ratios of
1:0, 1:0.1, 1:1, and 1:10 and reacted at room temperature for 10
minutes. Then, 50 .mu.l of the resulting reaction product was
placed on a plate coated with human TIGIT and incubated at room
temperature for 2 hours. The result was washed 3 times with PBST
(0.05% Tween 20 in PBS), and 50 .mu.l of anti-SA-HRP diluted 1:3000
with PBS was added thereto, followed by incubation at room
temperature for 1 hour. The result was washed 3 times with PBST
(0.05% Tween 20 in PBS) and 50 .mu.l of a TMB solution was added
thereto. The result was allowed to react at room temperature for 5
minutes, 50 .mu.l of a TMB stop solution was added thereto, and the
absorbance was measured at 450 nm in an ELISA leader.
[0130] The results are shown in FIG. 3. A blocking assay for TIGIT
and PVR was performed on 11 types of anti-TIGIT antibodies, and the
results of the assay showed that all 11 types of anti-TIGIT
antibodies blocked the ligand.
Example 4. Determination of Affinity of Anti-TIGIT Antibodies
[0131] 4.1 Determination Using Octet
[0132] A 5 .mu.g/ml of human TIGIT-HIS was prepared by diluting the
same in a 1.times.KB buffer. Anti-human TIGIT antibodies were
prepared through 1/2 serial dilution with 1.times.KB buffer from a
starting concentration of 100 nM. A penta-His sensor was reacted
with 1.times.KB buffer for 10 minutes, and the affinity of the
antibody to human TIGIT-HIS was measured using Octet. The results
are shown in Table 3 and FIG. 4.
TABLE-US-00005 TABLE 3 Sample ID KD (M) Full R{circumflex over (
)}2 kon(1/Ms) kdis(1/s) WIN-1B11 3.34E-10 0.9806 3.89E+05 1.30E-04
WIN-1B6 4.43E-10 0.9795 4.65E+05 2.06E-04 1G8 2.24E-10 0.9867
4.15E+05 9.32E-05 1F10 1.32E-09 0.9942 1.76E+05 2.32E-04 1F2
8.97E-10 0.9684 4.80E+05 4.31E-04 1E12 1.47E-09 0.9401 4.99E+05
7.32E-04 1E4 2.23E-10 0.9792 4.54E+05 1.01E-04 1C3 3.55E-09 0.9913
8.97E+04 3.18E-04 1E8 9.37E-10 0.9585 5.63E+05 5.28E-04 WIN-1D2
5.71E-10 0.975 4.88E+05 2.78E-04 1B11 3.16E-09 0.9576 3.21E+05
1.02E-03
[0133] The affinity of 11 anti-TIGIT antibodies was measured using
Octet and the result showed that among them, 1B11, 1E12, 1F2, and
1E8 had low dissociation compared to other clones.
[0134] 4.2 Conversion to IgG4-Type Anti-TIGIT Antibodies
[0135] The heavy-chain variable region and light-chain variable
region of each of the final five anti-human TIGIT antibodies were
synthesized.
TABLE-US-00006 TABLE 4 SEQ Clone ID name No. Heavy chain variable
region sequence 1F10 EVQLVESGGGLVKPGGSLRISCAAS 28
GFTFSNYNMIWVRQAPGKGLEWVSSI SSSASYIYYADSVKGRFTISRDNAKN
SLYLQMNSLRAEDTAVYYCAR DEGSRDSWNNGPYYYSGMDVWGQGTT VTVSS 1G8
EVQLVQSGAEVKKPGASVKVSCKAS 29 GYTFTSYYMHWVRQAPGQGLEWMGI
INPSGGSTSYAQKFQGRVTMTRDTS TSTVYMELSSLRSEDTAVYYC
ASRSGSGWFGALDYWGQGTLVTVSS WIN_1B QVQLVQSGAEVKKPGASVKVSCKAS 30 6
GYTFTSYGISWVRQAPGQGLEWMGW ISAYNGNTNYAQKLQGRVTMTTDTS
TSTAYMELRSLRSDDTAVYYC ARAGWEQQLGFDYWGQGTLVTVSS WIN_1D
QVQLVQSGAEVKKPGASVKVSCKAS 31 2 GYTFTSYGISWVRQAPGQGLEWMGW
ISAYNGNTNYAQKLQGRVTMTTDTS TSTAYMELRSLRSDDTAVYYC
ARAGWEQQLGFDYWGQGTLVTVSS 1E8 QVQLVQSGTEVKKPGASVKVSCKAS 32
GYTFSSYAITWVRQAPGQGLEWMGW ISAYNGNTNYAQKLQGRVTMTTDTS
TSTAYMELRSLRSDDTAVYYC ARVDFWSGYNYFDYWGQGTLVTVSS Ref-
EVQLQQSGPGLVKPSQTLSLTCAIS 33 erence GDSVSSNSAAWNWIRQSPSRGLEWL
GKTYYRFKWYSDYAVSVKGRITINP DTSKNQFSLQLNSVTPEDTAVFYCT
RESTTYDLLAGPFDYWGQGTLVTVSS Light chain variable region sequence
1F10 AIQMTQSPSSLSASVGDRVTITCRAS 34 QSISRYLNWYQHKPGKAPKLLIY
GASSLQSGVPSRFRGSGSGTDFTLTI SSLQPEDFATYYC QQSYTTPGAFTFGGGTKVEIKR 1G8
EIVLTQSPGTLSLSPGERATLSCRAS 35 QSVSSSYLAWYQQKPGQAPRLLIY
GASSRATGIPDRFSGSGSGTDFTLTI SRLEPEDFAVYYC QQYGSSPGGTFGQGTKVEIKR
WIN_1B AIQLTQSPSSLSASVGDRVTISCRAS 36 6 QTIRSYLNWYQQKRGKAPKLLIY
AASSLQSGVPLRFSGSGSGTDFTLTI SSLQPEDFATYYC QQSYSTLPLTFGGGTKVEIKR
WIN_1D AIQLTQSPSSLSASVGDRVTITCRAS 37 2 QSISSYLNWYQQKPGKAPKLLIY
AASSLQSGVPSRFSGSGSGTDFTLTI SSLQPEDFATYYC QQSYSTLAITFGQGTRLEIKR 1E8
DIQMTQSPSSLSASVGDRVTITCRAS 38 QGISNYLAWYQQKPGKVPKVLIY
AASTLQSGVPSRFSGSGSGTDFTLTI SSLQPEDVATYYC QKSNSAPLTFGGGTKVEIKR Ref-
DIVMTQSPDSLAVSLGERATINCKSS 39 erence QTVLYSSNNKKYLAWYQQKPGQP
PNLLIYWASTRESGVPDRFSGSGSGT DFTLTISSLQAEDVAVYYC
QQYYSTPFTFGPGTKVEIKR
TABLE-US-00007 TABLE 5 Clone name CDR1 CDR2 CDR3 1F10 GFTFSNYN
SSSASYI DEGSRDS VH (SEQ ID (SEQ ID (SEQ ID NO: 5) NO: 3) NO: 4) IF
10 QSISRY GASSLQS QQSYTTPGAFT VL (SEQ ID (SEQ ID (SEQ ID NO: 16)
NO: 14) NO: 15) 1G8 GYTFTSYY INPSGGST ASRSGSG VH (SEQ ID (SEQ ID
(SEQ ID NO: 8) NO: 6) NO: 7) IG8 QSVSSSY GASSRAT QQYGSSPGGT VL (SEQ
ID (SEQ ID (SEQ ID NO: 19) NO: 17) NO: 18) WIN_1B6 GYTFTSYG
ISAYNGNT ARAG VH (SEQ ID (SEQ ID (SEQ ID NO: 11) NO: 9) NO: 10)
WIN_1B6 QTIRSY AASSLQS QQSYSTLPLT VL (SEQ ID (SEQ ID (SEQ ID NO:
22) NO: 20) NO: 21) WIN_1D2 GYTFTSYG ISAYNGNT ARAG VH (SEQ ID (SEQ
ID (SEQ ID NO: 11) NO: 9) NO: 10) WIN_1D2 QSISSY AASSLQS QQSYSTLAIT
VL (SEQ ID (SEQ ID (SEQ ID NO: 24) NO: 23) NO: 21) 1E8 GYTFSSYA
ISAYNGNT ARVDF VH (SEQ ID (SEQ ID (SEQ ID NO: 13) NO: 12) NO: 10)
1E8 QGISNY AASTLQS QKSNSAPLT VL SEQ ID (SEQ ID (SEQ ID NO: 27) (NO:
25) NO: 26)
[0136] 4.3 Biacore Experiment
[0137] (1) Immobilization
[0138] 10 .mu.g/ml of anti-protein A was prepared by diluting the
same in an acetate 4.0 buffer. 100 nM NHS, 400 mM EDC and 1 M
ethanolamine were prepared. The sensor chip used herein was a CM5.
The contact time was set at 300s (5 min), the flow rate was set at
5 .mu.g/ml, and the immobilization level was set at 2,500 RU. After
performing running, whether the RU reached the target level was
monitored in the system control window.
[0139] (2) KD Measurement
[0140] The capture antibodies 1G8 and WIN 1B6 and a reference
antibody were diluted to 1 .mu.g/ml in 1.times.HBS-EP buffer, and
the target level was set to 300 RU. The antigen TIGIT-HIS was
subjected to 1/2 serial dilution from 160 nM with 1.times.HBS-EP
and reacted for a set contact time of 120 s and a set dissociation
time of 600 s. When running was completed, ka, kd, and KD were
obtained by fitting using evaluation software.
[0141] The results are shown in FIG. 5. Affinities of 1G8, WIN_1B6,
and the reference antibody were measured using BIACORE. The
reference antibody and candidate antibody 1G8 exhibited affinity of
nM or higher, whereas the other candidate antibody, WIN_1B6,
exhibited affinity of 10 nM or higher.
Example 5. Determination of Binding Capacity Between TIGIT
Expressed on the Cell Surface and TIGIT Candidate Antibody
[0142] In order to prepare a cell line stably expressing TIGIT,
Jurkat cells (Jurkat E6.1 (ATCC; TIB-152TM) were transfected with
TIGIT cDNA, treated with 1 mg/ml of antibiotic G418, and selected
to prepare a TIGIT-overexpressing cell line (Jurkat-TIGIT). The
Jurkat-TIGIT cell line was resuspended in DPBS supplemented with 2%
(v/v) FBS (hereinafter referred to as "FACS buffer") and
centrifuged at 1,500 rpm. The cells were resuspended in FACS buffer
so that the number of cells was 3.times.10.sup.6 cells/ml, and 100
.mu.l of the suspension was added to each well of a U-bottom
96-well plate. Then, the cells and culture solution of each well
were recovered and centrifuged at 1,500 rpm, and then the
supernatant was discarded. The recovered cells were resuspended in
50 .mu.l of FACS buffer supplemented with 0.5 .mu.l of a human Fc
block (BD Pharmingen; Cat. #564220) solution and incubated at
4.degree. C. for 15 minutes. The TIGIT candidate antibody or human
IgG4 (Sigma; Cat. #14639) was diluted in 50 .mu.l of FACS buffer at
the 2-time of the concentration 25 .mu.g/ml, 5 .mu.g/ml, 1
.mu.g/ml, 0.2 .mu.g/ml, 0.04 .mu.g/ml, 0.008 .mu.g/ml, 0.0016
.mu.g/ml, or 0.00032 .mu.g/ml. 50 .mu.l of the previously diluted
TIGIT candidate antibody or human IgG4 was added to the cells
containing an Fc blocker such that the concentration was adjusted
to each of 25 .mu.g/ml, 5 .mu.g/ml, 1 .mu.g/ml, 0.2 .mu.g/ml, 0.04
.mu.g/ml, 0.008 .mu.g/ml, 0.0016 .mu.g/ml, and 0.00032 .mu.g/ml and
reacted at 4.degree. C. for 1 hour 30 minutes. The cells reacted
with the TIGIT candidate antibody were resuspended in FACS buffer,
and then centrifuged at 1,500 rpm and washed. This series of
processes was repeated twice. Phycoerythrin (hereinafter, referred
to as "PE")-conjugated goat anti-human Fab'2 (Sigma; Cat. #P8047)
was diluted in FACS buffer at a volume ratio of 1:500, 100 .mu.l
thereof was added to each well, and the reaction was allowed to
proceed in the dark at 4.degree. C. for 30 minutes. The cells
reacted with PE were recovered, resuspended in FACS buffer, and
centrifuged at 1,500 rpm, the supernatant was discarded, and the
residue was washed. This series of processes was repeated twice.
Then, the cells were resuspended in 100 .mu.l of fixation buffer
(BD Cytofix.TM.; Cat. #554655) and incubated in the dark at
4.degree. C. for 30 minutes. The cells reacted with the fixation
buffer were recovered, resuspended in FACS buffer, and centrifuged
at 1,500 rpm, the supernatant was discarded, and the residue was
washed. This series of processes was repeated twice. The washed
cells were resuspended in 200 .mu.l of FACS buffer, and then the
median fluorescence intensity (MFI) of PE with which the cells were
labeled was compared using a FACS LSR-Fortessa instrument. All FACS
analysis was performed using FlowJo software. The results are shown
in FIG. 6. The results showed that WIN_1B6, WIN_1D2, 1E8, 1F10, and
1G8, among the TIGIT candidate antibodies, exhibited high binding
capacity.
Example 6. Determination of Competitiveness of TIGIT Candidate
Antibody for PVR, Ligand of TIGIT
[0143] The TIGIT-overexpressing cell line (Jurkat-TIGIT) was
resuspended in DPBS supplemented with 2% (v/v) FBS (hereinafter,
FACS buffer), centrifuged at 1,500 rpm, and resuspended in FACS
buffer so that the number of cells was 3.times.10.sup.6 cells/ml,
and 100 .mu.l of the suspension was added to each well of a
U-bottom 96-well plate. Then, the cells and culture solution of
each well were recovered and centrifuged at 1,500 rpm, and then the
supernatant was discarded. The recovered cells were resuspended in
50 .mu.l of FACS buffer supplemented with 0.5 .mu.l of a human Fc
block (BD Pharmingen; Cat. #564220) solution and incubated at
4.degree. C. for 15 minutes.
[0144] PVR-Fc purified from HEK293 cells (Thermo Fisher Scientific;
Cat. #A14527) was added in an amount of 10 .mu.g in 0.5 .mu.l to
each well and reacted at 4.degree. C. for 1 hour. The cells reacted
with PVR-Fc were resuspended in FACS buffer, centrifuged at 1,500
rpm, and washed. This series of processes was repeated twice. TIGIT
candidate antibody or human IgG4 (Sigma; Cat. #14639) was diluted
in 100 .mu.l of FACS buffer at concentrations of 10 .mu.g/ml, 0.2
.mu.g/ml, 0.04 .mu.g/ml, 0.008 .mu.g/ml, 0.0016 .mu.g/ml, and
0.00032 .mu.g/ml, 100 .mu.l thereof was added to each well, and the
cells were resuspended and then reacted at 4.degree. C. for 1 hour.
The cells reacted with the TIGIT candidate antibody were
resuspended in FACS buffer, centrifuged at 1,500 rpm, and washed.
This series of processes was repeated twice. Each well was treated
with an anti-PVR-PE antibody (Invitrogen; Cat. #12-1550-41) and
allowed to react in the dark at 4.degree. C. for 30 minutes. The
cells reacted with the anti-PVR-PE antibody were recovered,
resuspended in FACS buffer, and centrifuged at 1,500 rpm, the
supernatant was discarded and the residue was washed. This series
of processes was repeated twice. Then, the cells were resuspended
in 100 .mu.l of fixation buffer (BD Cytofix.TM.; Cat. #554655) and
incubated in the dark at 4.degree. C. for 30 minutes. The cells
reacted with the fixation buffer were recovered, resuspended in
FACS buffer, and centrifuged at 1,500 rpm, the supernatant was
discarded, and the residue was washed. This series of processes was
repeated twice. The washed cells were resuspended in 200 .mu.l of
FACS buffer. Then, the median fluorescence intensity (MFI) of PE
with which the cells were labeled was compared using a FACS
LSR-Fortessa instrument. All FACS analysis was performed using
FlowJo software. The results are shown in FIG. 7. The results
showed that among the TIGIT candidate antibodies, WIN_1B6, WUN
1B11, WIN_1D2, 1G8, and 1E8 strongly bound to PVR.
Example 7. Analysis of T-Cell Activation Due to Inhibitory Activity
of TIGIT Candidate Antibody on Binding Between TIGIT and PVR
[0145] T-cell activation by the TIGIT candidate antibody was
detected using the TIGIT/CD155 blockade bioassay kit (Promega, Cat
No. J2305). One vial of thaw-and-use TIGIT effector cells stored at
-140.degree. C. was thawed and resuspended in 12 ml of RPMI1640
(Gibco, Cat No. 11875-065) supplemented with 10% (v/v) FBS. 80
.mu.l of the cell solution was added into the inner 60 wells of a
white 96-well flat-bottom assay plate and incubated at 37.degree.
C. for 16 to 18 hours. The next day, one vial of thaw-and-use CD155
aAPC/CHO-K1 cells stored at -140.degree. C. was thawed and
resuspended in 3 ml of RPMI1640 (Gibco, Cat No. 11875-065)
supplemented with 10% (v/v) FBS. The TIGIT candidate antibody was
prepared at the highest concentration of 300 .mu.g/ml (6.times.),
and was serially diluted 10 times by 2.5.times. each time. 20 .mu.l
of an antibody was added to the TIGIT effector cell plate incubated
the previous day, 20 .mu.l of the thaw-and-use CD155 aAPC/CHO-K1
cell suspension was added thereto, and the result was incubated for
6 hours at 37.degree. C. in the presence of CO.sub.2 in an
incubator. After 6 hours, the 96-well plate was taken out from the
incubator and allowed to stand at room temperature for 20 minutes.
A Bio-Glo substrate was mixed with 10 ml of buffer, and each of the
60 wells containing the cell suspension was treated with 120 .mu.l
of the resulting mixture and allowed to stand for 10 minutes.
Luminescence was measured using the GloMax Discover System. The
results are shown in FIG. 8. The results showed that among the five
TIGIT candidate antibodies, upon treatment with 1G8, EC.sub.50 was
2.1 nM, which demonstrated that among the candidate antibodies, 1G8
exhibited the highest ability to activate T cells.
Example 8. Determination of Cross-Reaction of TIGIT Candidate
Antibody
[0146] In order to determine whether or not the TIGIT antibody has
binding ability to TIGIT of mice, monkeys and humans, each of
pEF1a-AcGFP-N1 vector (Clontech, Cat No. 631973) as a control
vector, mouse TIGIT, monkey TIGIT, and human TIGIT plasmid was
added in an amount of 20 .mu.g to HEK293 cells and then transfected
with Lipofectamine.TM. 3000 (Invitrogen, Cat No. L3000001). After
transfection for 48 hours, GFP expression was observed using a
fluorescence microscope, and the cells were detached by treatment
with 1 ml of TrypLE Express Solution (Thermo Fisher Scientific;
Cat. #12605010), and then diluted in 9 ml of DMEM (Gibco, Gibco,
Cat No. 11995-065) supplemented with 10% (v/v) FBS, and centrifuged
at 1,200 rpm for 5 minutes, and the supernatant was removed. The
result was washed once with PBS, and then the cells were
resuspended in FACS buffer to a concentration of 5.times.10.sup.5
cells/100 .mu.l. 1 .mu.l of a human Fc block solution was added to
each sample and reacted at 4.degree. C. for 10 minutes. The sample
was treated at a concentration of 1 .mu.g/5.times.10.sup.5 cells
with human IgG4, WIN_1B6, WIN_1D2, E8, 1F10, and 1G8, and reacted
at 4.degree. C. for 1 hour. The result was washed with FACS buffer,
PE-labeled goat-anti-human Fab'2 antibody was diluted at 1:200 in
FACS buffer, 100 .mu.l of the dilution was added to each sample,
and reaction was allowed to proceed at 4.degree. C. for 30 minutes.
The result was centrifuged in 200 .mu.l of FACS buffer at 1,200 rpm
for 5 minutes to remove the supernatant. The cells were resuspended
in 300 .mu.l of fixation buffer. The cells expressing GFP were
screened using a FACS LSR-Fortessa instrument, and TIGIT candidate
antibodies were identified using a PE fluorescence channel and
analyzed using FlowJo software. The results are shown in FIG. 9.
The results showed that, among the TIGIT candidate antibodies,
WIN_1B6, WIN_1D2, E8, and 1G8 bound to TIGIT of humans as well as
monkeys based on binding ability to proteins expressed on the cell
surface. However, the results showed that binding to TIGIT did not
occur in the case of mice.
Example 9. Determination of Inhibitory Activity of TIGIT Candidate
Antibody on Regulatory T Cells
[0147] 9.1 Isolation of Mononuclear Cells from Peripheral Blood
(PBMC)
[0148] Blood was transferred from a concentrated red blood cell bag
(200-250 mL) to a disposable bottle using an 18G needle and a 50 mL
syringe, and was diluted at a ratio of 1:1 in DPBS. 15 mL of
Ficoll-Paque medium (GE Healthcare, Cat No. 17-1440-03) was
prepared in a fresh 50 mL conical tube, and 30 mL of diluted blood
was slowly layered on the Ficoll-Paque medium by placing a pipette
tip on the wall of the conical tube. The result was centrifuged at
1,500 rpm and 25.degree. C. for 30 minutes (no brake). After
centrifugation, the PBMC layer was carefully collected and
transferred to a 50 ml fresh conical tube, a washing buffer was
added to adjust a total volume to 50 ml, and centrifugation was
performed at 1,500 rpm at 4.degree. C. for 10 minutes. The
supernatant was discarded, the cells were resuspended in a washing
buffer and centrifuged at 1,500 rpm and 4.degree. C. for 10
minutes, and the supernatant was discarded. Then, the result was
washed with IMDM (GIBCO, Cat No. 12440-) supplemented with 10%
(v/v) FBS 053), the number of cells and viability thereof were
determined, and the cells were incubated on a 6-well plate for 16
to 18 hours.
[0149] 9.2 Isolation of Regulatory T Cell
[0150] A regulatory T cell isolation kit (STEMCELL, Cat No. 18063)
was used to isolate regulatory T cells from PBMCs incubated the day
before. 5.times.10.sup.8 cells/ml of isolated human PBMCs were
placed in a round-bottom tube, 100 .mu.l of a CD25 positive
selection cocktail was added thereto, and then reaction was allowed
to proceed at room temperature for 5 minutes. 60 .mu.l of
releasable RapidSpheres (vortexed before use) was added thereto,
100 .mu.l of a CD4.sup.+ T cell enrichment cocktail was further
added thereto, and then reaction was allowed to proceed at room
temperature for 5 minutes. After resuspension, the round-bottom
tube was inserted into the magnet and reacted at room temperature
for 10 minutes. Then, the supernatant (CD4.sup.+ CD25.sup.-) was
carefully removed with a pipette and transferred to a fresh tube.
The tube was carefully separated from the magnet, 2.5 ml of EasySep
buffer was added thereto, and the result was carefully resuspended
with a pipette 2-3 times, placed on the magnet again, and reacted
at room temperature for 5 minutes, and then the supernatant
(CD4.sup.+ CD25.sup.-) was carefully removed with a pipette. This
series of processes was repeated twice. The tube was separated from
the magnet and EasySep buffer was added thereto in the same amount
as the initial amount, resuspension was performed 5 times or more
with 200 .mu.l of release buffer, 100 .mu.l of a CD127.sup.high
depletion cocktail was added thereto, and then the reaction was
allowed to proceed at room temperature for 5 minutes. 20 .mu.l of
Dextran RapidSpheres (vortexed before use) were added thereto,
followed by reaction at room temperature for 5 minutes and
resuspension. Then, a round-bottom tube was inserted into a magnet,
followed by reaction at room temperature for 5 minutes. The
supernatant (CD4.sup.+CD25.sup.+CD127.sup.low) was carefully
decanted with a pipette, transferred to a fresh tube, and washed
once with IMDM (GIBCO, Cat No. 12440-053) supplemented with 10%
(v/v) FBS. The number of cells was determined.
[0151] 9.3 Labeling Responder Cells with CFSE
[0152] 1.times.10.sup.6 cells/ml (1 ml) of the PBMCs incubated the
day before were placed in a 15 ml tube, 1 ml of 20 .mu.M CFSE
(eBioscience, Cat No. 65-0850-84) was added thereto, the cells were
mixed thoroughly, entry of light was blocked with silver foil, and
a reaction was allowed to proceed in a 37.degree. C. CO.sub.2
incubator for 20 minutes. After 20 minutes, cold IMDM supplemented
with 10 ml of 10% (v/v) FBS was added thereto, followed by
centrifugation at 1,200 rpm and 4.degree. C. for 5 minutes, and
this process was repeated twice.
[0153] 9.4 Co-Culture of Regulatory T Cells with CFSE-Labeled
Responder Cells
[0154] The CFSE-labeled responder cells were resuspended in IMDM
supplemented with 10% (v/v) FBS and seeded at 2.times.10.sup.5
cells/100 .mu.l on a 96-well round plate, and regulatory T cells
were added thereto at ratios of 0:1, 0.1:1 (2.times.10.sup.4),
0.25:1 (5.times.10.sup.4), and 0.5:1 (1.times.10.sup.5) (Treg:
responder). Soluble anti-CD3/anti-CD28 (2 .mu.g/ml) was added to
each well and human IgG4 and TIGIT antibody were added at a
concentration of 10 .mu.g/ml to each well, followed by incubation
for 5 days in a 37.degree. C. 5% CO.sub.2 incubator. After 5 days,
the cells and culture medium were recovered, centrifuged at 1,500
rpm, the supernatant was stored at -80.degree. C. for IFN-.gamma.
ELISA, the remaining cells were recovered, and Fc receptors were
blocked at 4.degree. C. in fresh FACS buffer for 15 minutes. The
plate was treated with antibodies to CD45-PE (TONBO, Cat No.
50-0459-T100), CD3-Percific blue (Biolegend, Cat No. 300330),
CD8-APC (TONBO, Cat No. 20-0088-T100), CD4-PE-Cy7 (TONBO, Cat No.
60-0049-T100), and CD25-APC-Cy7 (Biolegend, Cat No. 302614), each
being marked with a different dye, followed by reaction at
4.degree. C. for 30 minutes. After the reaction, a FACS buffer was
added and centrifugation was performed. This process was repeated
three times. The cells were resuspended in 100 .mu.l of fixation
buffer (BD Cytofix; cat. #554655) and reacted at 4.degree. C. for
30 minutes. The result was washed twice with FACS buffer and
resuspended in 200 .mu.l of FACS buffer for assay. Cell
proliferation was determined based on CFSE in CD8 cells using a
FACS LSR-Fortessa instrument and analyzed using FlowJo software.
The results are shown in FIG. 10. The results showed that the
function of regulatory T cells was suppressed by the TIGIT
candidate antibody and that the number of CD8 T cells was increased
by 14% compared to the control group.
[0155] 9.5 Detection of IFN-.gamma. Secretion by TIGIT Candidate
Antibody
[0156] The sample supernatant stored at -80.degree. C. was thawed
at room temperature and centrifuged at 1,500 rpm for 5 minutes. To
measure the amount of secreted IFN-.gamma., a human IFN-.gamma.
immunoassay kit (R&D SYSTEMS, Cat No. DIF50) was used. All
reagents used for ELISA were prepared at room temperature. As many
microplate strips as the number of samples were prepared, and 100
.mu.l of diluent RD1-51 was added to each well to which the sample
would be fed. The supernatant was diluted 20.times. with calibrator
diluent RD6-21. IFN-.gamma. standard stock (1,000 .mu.g/ml) was
diluted to 500 .mu.g/ml, and was then serially diluted 6 times at a
volume ratio of 1:1. 100 .mu.l of a standard and 100 .mu.l of a
sample were added to each well, followed by reaction at room
temperature for 2 hours. The result was washed 3 times with wash
buffer, and each well was treated with 200 .mu.l of a human
IFN-.gamma. conjugate, followed by reaction at room temperature for
2 hours. The result was washed 3 times with wash buffer, and each
well was treated with 200 .mu.l of a substrate solution, followed
by reaction at room temperature for 30 minutes. Each well was
treated with 50 .mu.l of a stop solution, and the O.D. value was
measured at a wavelength of 540 nm or 570 nm using molecular
dynamics reader equipment. The measured values were analyzed using
SoftMax Pro 5.4.1 program. The results are shown in FIG. 10. The
results showed that the function of regulatory T cells was
inhibited by the TIGIT candidate antibody and thus the amount of
secreted IFN-.gamma., measured in the culture supernatant, was
about two times higher than in the control group.
Example 10. Determination of Anticancer Activity of TIGIT Candidate
Antibody
[0157] Raji cancer cells and the Raji-PVR cell line prepared to
overexpress PVR were resuspended at a concentration of
1.times.10.sup.5 cells/100 .mu.l in RPMI 1640 medium (Thermo Fisher
Scientific; Cat. #11875093) in a 15 mL tube, 30 .mu.l of calcein-AM
(final 30 .mu.M, Invitrogen; Cat. #C3099) was added thereto,
followed by reaction in the dark at 37.degree. C. for 1 hour. After
1 hour, the result was washed twice with RPMI 1640 medium,
resuspended at a concentration of 1.times.10.sup.5 cells/100 .mu.l
in RPMI 1640 medium, and then added to a 96-well-plate. The
expanded NK cells were added to each well along with cancer cells
at a ratio of 3:1 thereto, and at this time, 1G8 was added to each
well at a concentration of 10 .mu.g/ml, and the result was treated
with human IgG4 as a control group in the same manner as above and
co-cultured in 5% CO.sub.2 in the dark at 37.degree. C. for 6
hours. After 6 hours, the 96-well-plate was centrifuged at 2,000
rpm for 3 minutes, 100 .mu.l of the supernatant was collected and
transferred to a 96-well-black plate, and the value of each sample
was measured at wavelengths of 485 nm/535 nm using a multiple
reader. The lysis value was assayed using the following formula.
The results are shown in FIG. 11. The results showed that cancer
cell apoptosis by NK cells was increased in PVR-overexpressing
cells on treatment with 1G8, and this is due to specific targeting
for TIGIT in a PVR-expression-dependent manner.
Lysis = Sample - Spon Max + ( MM - MT ) - Spon .times. 100
##EQU00001##
Example 11. Determination of Increase in T-Cell Reactivity Upon
Treatment with Combination of TIGIT Candidate Antibody (1G8) with
PD-1 Antibody
[0158] 11.1. Differentiation into Mature Dendritic Cells after
Isolation of CD14-Positive Cells from Mononuclear Cells Isolated
from Peripheral Blood (PBMC)
[0159] Cryopreserved healthy donor-derived peripheral blood
mononuclear cells (PBMCs) were rapidly thawed in a 37.degree. C.
water bath and transferred to a 50 mL conical tube, and a thawing
medium (RPMI, Gibco 11875-093+10% FBS, Gibco 16000-044) was added
dropwise thereto while shaking the same. Then, the supernatant was
removed by centrifugation at 1200 rpm at 4.degree. C. for 10
minutes, the residue was resuspended in 40 mL of MACS buffer
(PBS+0.5% FBS+2 mM EDTA), and the number of cells was counted. The
cells were treated at a concentration of 20 .mu.L/10.sup.7 cells
with CD14 microbeads (Miltenyi Biotec, 130-050-201), treated with
80 .mu.L of MACS buffer, and then incubated in the dark at
4.degree. C. for 15 minutes. Then, the supernatant was removed by
centrifugation at 1,350 rpm at 4.degree. C. for 8 minutes, and the
residue was resuspended in 500 .mu.L of MACS buffer and then loaded
on an LS column (Miltenyi Biotec 130-042-401) mounted on a
QuadroMACS separator (Miltenyi Biotec, 130-090-976). The LS column
was washed 3 times with 3 mL of MACS buffer, removed from the
QuadroMACS separator, transferred to a 15 mL conical tube, and
pressed with a plunger to obtain CD14-positive cells. The obtained
CD14-positive cells were resuspended at a concentration of
1.2.times.10.sup.6 cells/mL in complete RPMI (RPMI, Gibco
A10491-01+10% FBS+55 .mu.M .beta.-Mercaptoethanol, Gibco
21985-023+1.times. Antibiotic-Antimycotic, Gibco 15240) and mixed
with complete RPMI containing 2.times.GM-CSF (2.times.10.sup.3
U/mL) (R&D systems, 215-GM-010) and 2.times.IL-4
(2.times.10.sup.3 U/mL) (R&D systems, 204-IL-010) cytokines at
a ratio of 1:1, and the result was added to a 6-well plate and then
incubated in a CO.sub.2 incubator at 37.degree. C. After 3 days,
1.5 mL of the supernatant culture medium was removed from each well
using a 1 mL pipette, and 2 mL of complete RPMI medium containing
1.times.GM-CSF+1.times.IL-4 was added to each well. After 2 days, 2
mL of the culture solution was removed from each of the 6 wells and
3 mL of the medium containing each cytokine GM-CSF (1000 U/mL)+IL-4
(1000 U/mL)+TNF-.alpha. (10 ng/mL) (R&D systems,
210-TA-010)+IL-1.beta. (10 ng/mL) (R&D systems,
201-LB-005)+IL-6 (10 ng/mL) (R&D systems, 206-IL-010)+PGE2 (1
.mu.g/mL) (Sigma, P0409-1MG) was added thereto and the cells were
incubated in a CO.sub.2 incubator at 37.degree. C. for 2 days. On
the 7th day after releasing and incubating the cells, mature
dendritic cells adhered to the bottom were pipetted and collected
in a 50 mL conical tube, the number of cells was counted, and the
concentration thereof was adjusted to 1.times.10.sup.5
cells/mL.
[0160] 11.2. Isolation of Pan T Cells from Mononuclear Cells
Isolated from Peripheral Blood (PBMC) and VPD450 Staining
[0161] Cryopreserved peripheral blood mononuclear cells (PBMCs)
isolated from an allogeneic donor different from the donor from
whom dendritic cells were isolated were rapidly thawed in a
37.degree. C. water bath and transferred to a 50 mL conical tube,
and the thawed medium was added dropwise during mixing while
shaking. Then, the result was centrifuged at 1,200 rpm at 4.degree.
C. for 10 minutes to remove the supernatant and resuspended in 40
mL of MACS buffer, and the number of cells was counted. The cells
were treated at a density of 10 .mu.L/10.sup.7 cells with pan T
cell biotin antibodies (Miltenyi Biotec, 130-096-535), treated with
40 .mu.L of MACS buffer, and incubated in the dark at 4.degree. C.
for 5 minutes. Then, the cells were treated at a density of 20
.mu.L/10.sup.7 cells with anti-biotin microbeads (Miltenyi Biotec,
130-096-535), treated with 30 .mu.L of MACS buffer, and incubated
in the dark at 4.degree. C. for 10 minutes. After incubation, the
final volume was adjusted to 500 .mu.L with MACS buffer and then
the resulting cell solution was loaded onto the LS column mounted
on the QuadroMACS separator. The MACS buffer was loaded in an
amount of 3 mL three times to each LS column, all of the cells
released from the LS column were collected in a 15 mL conical tube,
and the number of cells was counted. The collected T cells were
centrifuged at 1,200 rpm at 4.degree. C. for 10 minutes, the
supernatant was removed, and then the residue was resuspended in
1.times.PBS solution to adjust the final concentration to
2.times.10.sup.7 cells/mL. A 1 .mu.M VPD450 solution having the
same volume as the solution containing the cells was thoroughly
mixed with the cells, light was blocked with aluminum foil, and the
reaction was allowed to proceed in a CO.sub.2 incubator at
37.degree. C. for 20 minutes. Then, 10 mL of thawing medium was
added, followed by centrifugation at 1,200 rpm at 4.degree. C. for
5 minutes, and the supernatant was removed. This series of
processes was repeated twice. The resulting T cells were
resuspended in complete RPMI medium to adjust the final
concentration to 2.times.10.sup.6 cells/mL.
[0162] 11.3. Mixed Lymphocyte Reaction (MLR) Test Using Dendritic
Cells and Allogeneic T Cells
[0163] The amount of complete RPMI medium was adjusted to
co-culture the dendritic cells matured in vitro for 7 days obtained
in 11.1 and the allogeneic T cells stained with VPD450 isolated in
2.2 at a ratio of 1:20, and the final concentration was adjusted as
follows (dendritic cell final concentration: 1.times.10.sup.5
cells/mL; final T-cell concentration: 2.times.10.sup.6 cells/mL).
5.times.10.sup.3 cells/50 .mu.L of dendritic cells and
1.times.10.sup.5 cells/50 .mu.L of T cells were transferred by
pipetting the same into each well of a 96-well U-bottom plate. The
cells were transferred at a constant density and co-cultured for 5
days under the five following conditions: 1) only dendritic cells
and T cells were cultured, 2) treatment with human IgG4 type
isotype antibody (BioLegend, 403702), in addition to condition #1,
3) treatment with TIGIT candidate antibody (1G8) alone, in addition
to condition #1, 4) treatment with PD-1 antibody (MSD,
Pembrolizumab) alone, in addition to condition #1, and 5) treatment
with a combination of candidate antibody (1G8) and PD-1 antibody
TIGIT, in addition to condition #1. In all experiments, the final
concentration of each antibody was adjusted to be 5 .mu.g/mL. After
treatment with each antibody, the 96-well plate was incubated in a
CO.sub.2 incubator at 37.degree. C. for 5 days. After 5 days, the
cells and culture solution were recovered and centrifuged at 2,000
rpm, and the supernatant was stored in a -80.degree. C. freezer for
human IFN-.gamma. ELISA. FACS buffer (1% FBS/sheath buffer) was
added to the cells remaining in the 96-well plate, the result was
centrifuged at 1,200 rpm and 4.degree. C. for 5 minutes, and the
supernatant was removed. This series of processes was repeated
twice. The residue was resuspended in 100 .mu.L of FACS buffer,
transferred to a 5 mL tube, treated with antibodies, and incubated
in the dark at 4.degree. C. for 30 minutes. The antibodies used
herein were FITC anti-CD8 (BioLegend 301006), PE anti-TIGIT
(eBiosciences, 12-9500-42), PE-Cy7 anti-PD-1 (eBiosciences,
25-2799-42), APC anti-CD4 (Tonbo 20-0049-T100), APC-Cy7 anti-CD3
(BD 560176), and PerCP Cy5.5 anti-7AAD (BD 559925). Then, 1 mL of
FACS buffer was added to each sample, followed by centrifugation at
2,000 rpm at 4.degree. C. for 3 minutes, and then the supernatant
was removed to thereby obtain a sample. The cell proliferation
level of each group was determined and analyzed through the VPD450
negative/positive proportions in CD4-positive T cells and
CD8-positive T cells using LSR Fortessa. The results are shown in
FIG. 12a.
[0164] As can be seen from FIG. 12a, the TIGIT candidate antibody
(1G8)-treated group did not exhibit a significant increase in cell
proliferation in both CD4-positive T cells and CD8-positive T cells
compared to the IgG4 isotype antibody-treated control group,
whereas the PD-1 antibody-treated group exhibited a statistically
significant increase in cell proliferation. At this time,
statistically significant, further increased cell proliferation was
observed upon treatment with a combination of the PD-1 antibody and
the TIGIT candidate antibody (1G8), compared to treatment with the
PD-1 antibody alone.
[0165] 11.4. Observation of Change in IFN-.gamma. Secretion
[0166] The sample supernatant stored at -80.degree. C. was thawed
at room temperature and centrifuged at 1,500 rpm for 5 minutes.
Human IFN-gamma Quantikine ELISA Kit (R&D SYSTEMS, DIF50) was
used to measure the amount of IFN-.gamma. that was secreted. All
reagents used for ELISA were prepared at room temperature. As many
microplate strips as the number of samples were prepared, and 100
.mu.l of diluent RD1-51 was added to each well to which the sample
would be fed. The supernatant was diluted 20.times. with calibrator
diluent RD6-21. IFN-.gamma. standard stock (1,000 .mu.g/ml) was
diluted to 500 .mu.g/ml, and was then serially diluted 6 times at a
volume ratio of 1:1. 100 .mu.l of a standard and 100 .mu.l of a
sample were added to each well, followed by reaction at room
temperature for 2 hours. The result was washed 3 times with wash
buffer, and each well was treated with 200 .mu.l of a human
IFN-.gamma. conjugate, followed by reaction at room temperature for
2 hours. The result was washed 3 times with wash buffer, and each
well was treated with 200 .mu.l of a substrate solution, followed
by reaction at room temperature for 30 minutes. Each well was
treated with 50 .mu.l of the stop solution, and the O.D. value was
measured at a wavelength of 540 nm or 570 nm using molecular
dynamics reader equipment. The measured values were analyzed using
SoftMax Pro 5.4.1 program. The results are shown in FIG. 12b.
[0167] As can be seen from FIG. 12b, there was no difference in the
amount of IFN-.gamma. secretion between the TIGIT candidate
antibody-treated group and the control group, namely, the IgG4
isotype antibody-treated group, whereas there was a statistically
significant increase in the amount of IFN-.gamma. secretion between
the PD-1 antibody-treated group and the control group. At this
time, statistically significant, further increased (47% in average)
IFN-.gamma. secretion was observed upon treatment with a
combination of the PD-1 antibody with the TIGIT candidate antibody
(1G8), compared to treatment with the PD-1 antibody alone.
Example 12. Determination of Reduction of Regulatory T Cell
Activation Marker Expression by TIGIT Candidate Antibody (1G8)
[0168] 12.1. Treatment of Mononuclear Cells Isolated from
Peripheral Blood (PBMCs) of Healthy Donor with TIGIT Candidate
Antibody (1G8)
[0169] Cryopreserved healthy donor-derived peripheral blood
mononuclear cells were rapidly thawed in a 37.degree. C. water
bath, then transferred to a 50 mL conical tube, and the thawing
medium was added dropwise by mixing while shaking. Then, the result
was centrifuged at 1,200 rpm at 4.degree. C. for 10 minutes to
remove the supernatant, the cells were resuspended in 15 mL of a
thawing medium, and the number of cells was counted. The cells were
resuspended at 1.times.10.sup.7 cells/mL in complete RPMI,
1.times.10.sup.6 cells and 2 .mu.L of Dynabeads human T-activator
CD3/CD28 (gibco, 111.31D) were seeded to a total of 200 .mu.L/well
in 3 times on a 96-well U-bottom plate. Each group was treated with
the corresponding antibody (1G8 or hIgG4) at a final concentration
of 1 mg/mL and incubated for 6 days.
[0170] 12.2. Analysis of Transformation of Regulatory T Cells
[0171] The plate containing the cells incubated for 6 days was
centrifuged at 2,000 rpm and at 4.degree. C. for 3 minutes to
remove the supernatant, resuspended in 200 .mu.L of PBS per well,
and centrifuged at 2,000 rpm and at 4.degree. C. for 3 minutes.
After removing the supernatant, in order to isolate dead cells, the
residue was treated with LIVE/DEAD.TM. Fixable Aqua Dead Cell Stain
Kit (Invitrogen, L34957) and incubated in the dark at 4.degree. C.
for 30 minutes. Each well was further treated with 100 .mu.L of
FACS buffer and centrifuged at 2,000 rpm at 4.degree. C. for 3
minutes. The supernatant was removed and the residue was treated
with antibodies and incubated in the dark at 4.degree. C. for 3
minutes for 30 minutes. The antibodies herein used were BV421
anti-CD25 (BD 562442), FITC anti-CD127 (BD 564423), BB700 anti-CD8
(BD 566452), R700 anti-CD4 (BD 564975), APC-H7 anti-CD3 (BD
560176), PE-Cy7 anti-CD39 (eBioscience 25-0399-41), PE-Cy7
anti-PD-1 (Biolegend 135216), Alexa647 anti-ICOS (Biolegend
313516), Alexa647 anti-TIGIT (Biolegend 372724), PE-Cy7 anti-LAG-3
(Biolegend 369310), PE-Cy7 anti-HLA-DR (BD 565096), and APC
anti-CCM1 (R&D FAB2244A). Each well was further treated with
100 .mu.L of FACS and centrifuged at 2,000 rpm at 4.degree. C. for
3 minutes. The supernatant was removed and intracellular staining
was performed in accordance with the protocol of
FoxP3/transcription factor staining buffer set (eBioscience,
00-5523-00). The antibodies used herein were PE anti-FoxP3
(eBiosicence 12-4776-42) and APC anti-CTLA-4 (R&D FAB386A). The
results of an assay of stained cells using LSR Fortessa are shown
in FIG. 13.
[0172] As can be seen from FIG. 13, among the activation markers of
regulatory T cells of a normal subject, the 1G8 antibody did not
affect the expression of ICOS, PD-1, or LAG3, but significantly
reduced the expression of CD39 and CTLA-4.
Example 13. Analysis of Exhaustion Maker Expression of CD8 T Cells
and Regulatory T Cells in Patients with Multiple Myeloma
[0173] Cryopreserved peripheral blood mononuclear cells derived
from multiple myeloma patients were rapidly thawed in a 37.degree.
C. water bath, transferred to a 50 mL conical tube, and mixed
dropwise with the thawing medium while shaking. Then, the cells
were centrifuged at 1,200 rpm at 4.degree. C. for 10 minutes, the
supernatant was removed, the cells were resuspended in 15 mL of a
thawing medium, and the number of cells was counted. The thawed
peripheral blood mononuclear cells were aliquoted into four FACS
tubes, 2 mL of PBS was added thereto, and the result was
centrifuged at 4.degree. C. at 2,000 rpm for 3 minutes. The
supernatant was removed, and in order to isolate dead cells, the
residue was treated with LIVE/DEAD.TM. Fixable Aqua Dead Cell Stain
Kit and incubated at 4.degree. C. in the dark for 30 minutes. Each
tube was further treated with 2 mL of FACS buffer and centrifuged
at 4.degree. C. at 2,000 rpm for 3 minutes. After removing the
supernatant and treating with antibodies, the cells were incubated
in the dark for 30 minutes at 4.degree. C. The antibodies used
herein were Alexa700 anti-CD3 (BD 557943), PerCP-Cy5.5 anti-CD8
(Biolegend 344710), BB700 anti-CD8 (BD 566452), R700 anti-CD4 (BD
564975), BV421 anti-CD25 (BD 562442), BV786 anti-CD127 (BD 563324),
FITC anti-CD138 (Biolegend 356508), PE-Cy7 anti-PD-(Biolegend
135216), BV421 anti-TIGIT (BD 747844), Alx647 anti-TIGIT (Biolegend
372724), APC anti-Tim-3 (R&D FAB2356aA), PE-Cy7 anti-LAG-3
(Biolegend 369310), and APC anti-CTLA-4 (R&D FAB386A). After
staining, 2 mL of FACS buffer was added to each tube, and the cells
were centrifuged at 4.degree. C. at 2,000 rpm for 3 minutes. The
supernatant was removed and intracellular staining was performed in
accordance with the protocol of the FoxP3/transcription factor
staining buffer set. The antibodies used herein were PE anti-FoxP3
(eBiosicence 12-4776-42) and APC anti-CTLA-4 (R&D FAB386A). The
results of analysis of stained cells with LSR Fortessa are shown in
FIG. 14.
[0174] As can be seen from FIG. 14, the expression of TIGIT among
the five exhaustion markers of CD8 T cells in patients with
multiple myeloma was significantly higher than that of the other
four types. TIGIT was expressed in higher levels in regulatory T
cells from multiple myeloma patients, compared to PD-1.
Example 14. Determination of IFN-.gamma. Expression Increase in
Peripheral Blood Mononuclear Cells of Multiple Myeloma Patients by
TIGIT Candidate Antibody (1G8)
[0175] 14.1 Treatment of Peripheral Blood Mononuclear Cells of
Multiple Myeloma Patients with TIGIT Candidate Antibody (1G8)
[0176] Cryopreserved peripheral blood mononuclear cells derived
from multiple myeloma patients were rapidly thawed in a 37.degree.
C. water bath and transferred to a 50 mL conical tube, and a
thawing medium was added dropwise by mixing while shaking. Then,
the cells were centrifuged at 1,200 rpm at 4.degree. C. for 10
minutes, the supernatant was removed, the cells were resuspended in
15 mL of a thawing medium, and the number of cells was counted. The
cells were resuspended at 5.times.10.sup.6 cells/mL in complete
RPMI, 1.times.10.sup.5 cells/well and 2 .mu.L of Dynabeads human
T-activator CD3/CD28 were seeded 3 times on a 96-well U-bottom
plate. Each group was treated at 1 mg/mL with the corresponding
antibodies (TIGIT candidate antibody (1G8), PD-1 antibody, hIgG4, a
combination of TIGIT candidate antibody (1G8) and PD-1 antibody).
After 4 days, the result was centrifuged at 2,000 rpm at 4.degree.
C. for 3 minutes, and the supernatant culture solution was
collected at 100 .mu.L in a 96-well U-bottom plate and sealed and
stored at -20.degree. C.
[0177] 14.2 Determination of Change in IFN-.gamma. Secretion
[0178] The culture medium stored at -20.degree. C. was allowed to
thaw on the plate at room temperature. The amount of IFN-.gamma.
secretion was measured in accordance with the protocol of the BDTM
cytometric bead array (CBA) human Th1/Th2/Th17 cytokine kit (BD,
560484). The lyophilized cytokine standard stock provided in the
kit was transferred to a 15 mL tube, thawed by sufficiently
pipetting with 2 mL of a diluent buffer, and then was allowed to
stand at room temperature for at least 15 minutes. Then, the result
was serially diluted at 1:1 with 300 .mu.L of the same volume of
buffer to prepare ten standards. A negative reference standard was
prepared as a diluent buffer. The culture medium was diluted 1:20
using the diluent buffer of the kit. As many capture beads of each
cytokine as the number of samples and standards were prepared, and
50 .mu.L of the sample, 50 .mu.L of the mixed capture bead sample,
and 50 .mu.L of the PE detection antibody were added to 96-well 1.1
mL Cluster Tubes Bulk (Axygen, MTS-11-C), followed by thorough
mixing. The result was incubated in the dark for 3 hours, 300 .mu.L
of wash buffer was added thereto, centrifugation was performed at
4.degree. C. at 2,000 rpm for 3 minutes, and the supernatant was
removed. The IFN-.gamma. secretion of each sample after the process
was assayed using LSR Fortessa and the result is shown in FIG.
15.
[0179] As can be seen from FIG. 15, the amount of IFN-.gamma. that
was secreted when anti-CD3/28 stimulation was applied to the PBMCs
of multiple myeloma patients was significantly increased upon
treatment with the TIGIT candidate antibody (1G8).
Example 15. Determination of Tumor Growth Inhibitory Activity in
Human Colorectal Cancer Cell Xenograft Model Using Humanized Mice
by TIGIT Candidate Antibody (1G8)
[0180] In order to detect the tumor growth inhibitory effect of the
TIGIT candidate antibody (1G8) in vivo, a xenograft animal model in
which peripheral blood mononuclear cells from normal subjects and
the human cell line were transplanted into immune-cell-knocked-out
NOG (NOD/Shi-scid/IL-2R.gamma.null, Jackson Laboratory, 5-6 weeks
old) mice was used. The human cancer cell line used herein was a
human colon cancer cell line HT29 (ATCC, HTB-38), PVR expression
was detected using a PE anti-PVR antibody (Thermo Fisher
Scientific, 12-1550-41), and the results are shown in FIG. 16. PVR
expressed in HT29 cells bound to TIGIT expressed in immune cells to
induce activity of inhibition of immune cell activity. First,
3.5.times.10.sup.6 cells/0.2 mL of HT29 cells were injected
subcutaneously under the right foreleg of female NOG mice to induce
tumor formation (Day 0). Six hours after tumor cell injection,
Peripheral blood mononuclear cells were injected intraperitoneally
at 7.times.10.sup.6 cells/0.2 mL to create a mouse model having a
human immune system. When the tumor volume grew to 50-80 mm.sup.3,
the tumor was classified into a group having a similar tumor size,
and the TIGIT candidate antibody (1G8) (10 mg/kg) was administered
intraperitoneally to mice twice a week, a total of 6 times. The
average tumor volume of such an experimental group administered
with the TIGIT candidate antibody (1G8) at a dose of 10 mg/kg
exhibited tumor growth inhibitory activity of about 28.1% compared
to the average tumor volume of the negative control group (*:
P<0.05).
INDUSTRIAL APPLICABILITY
[0181] It was found that the anti-TIGIT antibody or antigen-binding
fragment thereof according to the present invention is specific to
TIGIT, strongly binds thereto, and exhibits superior therapeutic
efficacy compared to conventional anti-TIGIT antibodies. Therefore,
the anti-TIGIT antibody or antigen-binding fragment thereof
according to the present invention can be used as an
immuno-oncology agent through immune cell activation.
[0182] In addition, the anti-TIGIT antibody or antigen-binding
fragment thereof of the present invention can be used in
combination therapy with chemical drugs and other anticancer
drugs.
[0183] Although specific configurations of the present invention
have been described in detail, those skilled in the art will
appreciate that this description is provided to set forth preferred
embodiments for illustrative purposes, and should not be construed
as limiting the scope of the present invention. Therefore, the
substantial scope of the present invention is defined by the
accompanying claims and equivalents thereto.
SEQUENCE LISTING FREE TEXT
[0184] An electronic file is attached.
Sequence CWU 1
1
391244PRTArtificial SequenceSynthetic Construct 1Met Gly Trp Cys
Leu Leu Leu Ile Trp Ala Gln Gly Leu Arg Gln Ala1 5 10 15Pro Leu Ala
Ser Gly Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn 20 25 30Ile Ser
Ala Glu Lys Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser 35 40 45Ser
Thr Thr Ala Gln Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln 50 55
60Leu Leu Ala Ile Cys Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser65
70 75 80Phe Lys Asp Arg Val Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu
Gln 85 90 95Ser Leu Thr Val Asn Asp Ala Gly Glu Tyr Phe Cys Ile Tyr
His Thr 100 105 110Tyr Pro Asp Gly Thr Tyr Thr Gly Arg Ile Phe Leu
Glu Val Leu Glu 115 120 125Ser Ser Val Ala Glu His Gly Ala Arg Phe
Gln Ile Pro Leu Leu Gly 130 135 140Ala Met Ala Ala Thr Leu Val Val
Ile Cys Thr Ala Val Ile Val Val145 150 155 160Val Ala Leu Thr Arg
Lys Lys Lys Ala Leu Arg Ile His Ser Val Glu 165 170 175Gly Asp Leu
Arg Arg Lys Ser Ala Gly Gln Glu Glu Trp Ser Pro Ser 180 185 190Ala
Pro Ser Pro Pro Gly Ser Cys Val Gln Ala Glu Ala Ala Pro Ala 195 200
205Gly Leu Cys Gly Glu Gln Arg Gly Glu Asp Cys Ala Glu Leu His Asp
210 215 220Tyr Phe Asn Val Leu Ser Tyr Arg Ser Leu Gly Asn Cys Ser
Phe Phe225 230 235 240Thr Glu Thr Gly2317PRTArtificial
SequenceSynthetic Construct 2Gly Asp Val Val Val Gln Ala Pro Thr
Gln Val Pro Gly Phe Leu Gly1 5 10 15Asp Ser Val Thr Leu Pro Cys Tyr
Leu Gln Val Pro Asn Met Glu Val 20 25 30Thr His Val Ser Gln Leu Thr
Trp Ala Arg His Gly Glu Ser Gly Ser 35 40 45Met Ala Val Phe His Gln
Thr Gln Gly Pro Ser Tyr Ser Glu Ser Lys 50 55 60Arg Leu Glu Phe Val
Ala Ala Arg Leu Gly Ala Glu Leu Arg Asn Ala65 70 75 80Ser Leu Arg
Met Phe Gly Leu Arg Val Glu Asp Glu Gly Asn Tyr Thr 85 90 95Cys Leu
Phe Val Thr Phe Pro Gln Gly Ser Arg Ser Val Asp Ile Trp 100 105
110Leu Arg Val Leu Ala Lys Pro Gln Asn Thr Ala Glu Val Gln Lys Val
115 120 125Gln Leu Thr Gly Glu Pro Val Pro Met Ala Arg Cys Val Ser
Thr Gly 130 135 140Gly Arg Pro Pro Ala Gln Ile Thr Trp His Ser Asp
Leu Gly Gly Met145 150 155 160Pro Asn Thr Ser Gln Val Pro Gly Phe
Leu Ser Gly Thr Val Thr Val 165 170 175Thr Ser Leu Trp Ile Leu Val
Pro Ser Ser Gln Val Asp Gly Lys Asn 180 185 190Val Thr Cys Lys Val
Glu His Glu Ser Phe Glu Lys Pro Gln Leu Leu 195 200 205Thr Val Asn
Leu Thr Val Tyr Tyr Pro Pro Glu Val Ser Ile Ser Gly 210 215 220Tyr
Asp Asn Asn Trp Tyr Leu Gly Gln Asn Glu Ala Thr Leu Thr Cys225 230
235 240Asp Ala Arg Ser Asn Pro Glu Pro Thr Gly Tyr Asn Trp Ser Thr
Thr 245 250 255Met Gly Pro Leu Pro Pro Phe Ala Val Ala Gln Gly Ala
Gln Leu Leu 260 265 270Ile Arg Pro Val Asp Lys Pro Ile Asn Thr Thr
Leu Ile Cys Asn Val 275 280 285Thr Asn Ala Leu Gly Ala Arg Gln Ala
Glu Leu Thr Val Gln Val Lys 290 295 300Glu Gly Pro Pro Ser Glu His
Ser Gly Met Ser Arg Asn305 310 31538PRTArtificial SequenceSynthetic
Construct 3Gly Phe Thr Phe Ser Asn Tyr Asn1 547PRTArtificial
SequenceSynthetic Construct 4Ser Ser Ser Ala Ser Tyr Ile1
557PRTArtificial SequenceSynthetic Construct 5Asp Glu Gly Ser Arg
Asp Ser1 568PRTArtificial SequenceSynthetic Construct 6Gly Tyr Thr
Phe Thr Ser Tyr Tyr1 578PRTArtificial SequenceSynthetic Construct
7Ile Asn Pro Ser Gly Gly Ser Thr1 587PRTArtificial
SequenceSynthetic Construct 8Ala Ser Arg Ser Gly Ser Gly1
598PRTArtificial SequenceSynthetic Construct 9Gly Tyr Thr Phe Thr
Ser Tyr Gly1 5108PRTArtificial SequenceSynthetic Construct 10Ile
Ser Ala Tyr Asn Gly Asn Thr1 5114PRTArtificial SequenceSynthetic
Construct 11Ala Arg Ala Gly1128PRTArtificial SequenceSynthetic
Construct 12Gly Tyr Thr Phe Ser Ser Tyr Ala1 5135PRTArtificial
SequenceSynthetic Construct 13Ala Arg Val Asp Phe1
5146PRTArtificial SequenceSynthetic Construct 14Gln Ser Ile Ser Arg
Tyr1 5157PRTArtificial SequenceSynthetic Construct 15Gly Ala Ser
Ser Leu Gln Ser1 51611PRTArtificial SequenceSynthetic Construct
16Gln Gln Ser Tyr Thr Thr Pro Gly Ala Phe Thr1 5 10177PRTArtificial
SequenceSynthetic Construct 17Gln Ser Val Ser Ser Ser Tyr1
5187PRTArtificial SequenceSynthetic Construct 18Gly Ala Ser Ser Arg
Ala Thr1 51910PRTArtificial SequenceSynthetic Construct 19Gln Gln
Tyr Gly Ser Ser Pro Gly Gly Thr1 5 10206PRTArtificial
SequenceSynthetic Construct 20Gln Thr Ile Arg Ser Tyr1
5217PRTArtificial SequenceSynthetic Construct 21Ala Ala Ser Ser Leu
Gln Ser1 52210PRTArtificial SequenceSynthetic Construct 22Gln Gln
Ser Tyr Ser Thr Leu Pro Leu Thr1 5 10236PRTArtificial
SequenceSynthetic Construct 23Gln Ser Ile Ser Ser Tyr1
52410PRTArtificial SequenceSynthetic Construct 24Gln Gln Ser Tyr
Ser Thr Leu Ala Ile Thr1 5 10256PRTArtificial SequenceSynthetic
Construct 25Gln Gly Ile Ser Asn Tyr1 5267PRTArtificial
SequenceSynthetic Construct 26Ala Ala Ser Thr Leu Gln Ser1
5279PRTArtificial SequenceSynthetic Construct 27Gln Lys Ser Asn Ser
Ala Pro Leu Thr1 528129PRTArtificial SequenceSynthetic Construct
28Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1
5 10 15Ser Leu Arg Ile Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn
Tyr 20 25 30Asn Met Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Ser Ile Ser Ser Ser Ala Ser Tyr Ile Tyr Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Glu Gly Ser Arg Asp Ser
Trp Asn Asn Gly Pro Tyr Tyr 100 105 110Tyr Ser Gly Met Asp Val Trp
Gly Gln Gly Thr Thr Val Thr Val Ser 115 120
125Ser29121PRTArtificial SequenceSynthetic Construct 29Glu Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Tyr
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe
50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val
Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Ser Arg Ser Gly Ser Gly Trp Phe Gly Ala Leu
Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser 115
12030120PRTArtificial SequenceSynthetic Construct 30Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Gly Ile
Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly
Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55
60Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr65
70 75 80Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Ala Gly Trp Glu Gln Gln Leu Gly Phe Asp Tyr Trp
Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115
12031120PRTArtificial SequenceSynthetic Construct 31Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Gly Ile
Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly
Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55
60Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr65
70 75 80Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Ala Gly Trp Glu Gln Gln Leu Gly Phe Asp Tyr Trp
Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115
12032121PRTArtificial SequenceSynthetic Construct 32Gln Val Gln Leu
Val Gln Ser Gly Thr Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr 20 25 30Ala Ile
Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly
Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55
60Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr65
70 75 80Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Val Asp Phe Trp Ser Gly Tyr Asn Tyr Phe Asp Tyr
Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser 115
12033126PRTArtificial SequenceSynthetic Construct 33Glu Val Gln Leu
Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10 15Thr Leu Ser
Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25 30Ser Ala
Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45Trp
Leu Gly Lys Thr Tyr Tyr Arg Phe Lys Trp Tyr Ser Asp Tyr Ala 50 55
60Val Ser Val Lys Gly Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn65
70 75 80Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala
Val 85 90 95Phe Tyr Cys Thr Arg Glu Ser Thr Thr Tyr Asp Leu Leu Ala
Gly Pro 100 105 110Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser 115 120 12534110PRTArtificial SequenceSynthetic Construct
34Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Arg
Tyr 20 25 30Leu Asn Trp Tyr Gln His Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Gly Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg
Phe Arg Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Ser Tyr Thr Thr Pro Gly 85 90 95Ala Phe Thr Phe Gly Gly Gly Thr Lys
Val Glu Ile Lys Arg 100 105 11035110PRTArtificial SequenceSynthetic
Construct 35Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser
Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val
Ser Ser Ser 20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala
Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile
Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr
Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95Gly Gly Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg 100 105 11036109PRTArtificial
SequenceSynthetic Construct 36Ala Ile Gln Leu Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Ser Cys Arg
Ala Ser Gln Thr Ile Arg Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu
Gln Ser Gly Val Pro Leu Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Leu Pro 85 90 95Leu Thr
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100
10537109PRTArtificial SequenceSynthetic Construct 37Ala Ile Gln Leu
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu Asn
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr
Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Leu
Ala 85 90 95Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg 100
10538108PRTArtificial SequenceSynthetic Construct 38Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn Tyr 20 25 30Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Val Leu Ile 35 40 45Tyr
Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys Ser Asn Ser Ala Pro
Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100
10539114PRTArtificial SequenceSynthetic Construct 39Asp Ile Val Met
Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala
Thr Ile Asn Cys Lys Ser Ser Gln Thr Val Leu Tyr Ser 20 25 30Ser Asn
Asn Lys Lys Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45Pro
Pro Asn Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55
60Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65
70 75 80Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln
Gln 85 90 95Tyr Tyr Ser Thr Pro Phe Thr Phe Gly Pro Gly Thr Lys Val
Glu Ile 100 105 110Lys Arg
* * * * *
References